Methods of inducing tissue regeneration

ABSTRACT

Methods are provided for producing cells within a lineage (lineage restricted cells) from post-mitotic differentiated cells of the same lineage ex vivo and in vivo, and for treating a subject in need of tissue regeneration therapy by employing these lineage-restricted cells. In addition, the production of lineage restricted cells from postmitotic tissues derived from patients with diseases allows for a characterization of pathways that have gone awry in these diseases and for screening of drugs that will ameliorate or correct the defects as a means of novel drug discovery. Also provided are kits for performing these methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/394,798 filed on Mar. 14, 2012, which is a 371 U.S. National Phase ofApplication No. PCT/US10/57102 filed on Nov. 17, 2010, which claimspriority to the filing date of the U.S. Provisional Patent ApplicationSer. No. 61/281,575 filed Nov. 18, 2009; the disclosure of which areherein incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under contractsAG009521, AG020961, HD007249, and AR051678 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention pertains to methods for inducing tissue regeneration byproducing cells within a lineage, i.e. lineage-restricted cells, frompost-mitotic differentiated cells of the same lineage ex vivo and invivo. Also provided are kits for performing these methods.

BACKGROUND OF THE INVENTION

Tissue regeneration in humans is extremely limited and constitutes amajor challenge to the repair of damaged organ function. A number oforgans rely on undifferentiated stem and progenitor cells for tissueregeneration. However, it is unclear if resident stem cells are capableof regenerating the full mass of tissue required for a given injury.Furthermore, stem cells have not yet been identified for a number oftissues, and in those tissues in which stem cells have been identified,the factors required to induce their propagation and differentiation toacquire the fates of cells in these tissues are not fully understood.Thus, there is a need for methods of inducing well defineddifferentiated cells of known identity to contribute to cell replacementand tissue regeneration in vivo. Moreover, propagation of such cells exvivo can be used as cell based therapies upon delivery in vivo. Suchmethods are also applicable to modeling human diseases ex vivo (eg.skeletal, neuronal, cardiac, pancreatic, hepatic diseases and the like)and elucidating the underlying defects. In addition drugs capable ofameliorating the human disease phenotype can be screened using suchdisease models.

SUMMARY OF THE INVENTION

Methods for producing cells within a lineage, i.e. lineage-restrictedcells (LRCs), from post-mitotic differentiated cells (PMDs) of the samelineage ex vivo and in vivo are provided, wherein the lineage-restrictedcells may encompass mitotic progenitor cells committed to a cell lineage(MPC), post-mitotic immature cells committed to a particular type ofcell in the cell lineage (post-mitotic immature cell, PMI), andpost-mitotic differentiated cells of the cell lineage (post-mitoticdifferentiated cell, PMD). Also provided are methods for treating asubject in need of tissue regeneration therapy by employing cellsproduced by methods of the invention. Also provided are kits forperforming these methods.

In some embodiments of the invention, a post-mitotic differentiated cell(PMD) is contacted ex vivo with an effective amount of an agent thattransiently inhibits activity of a member of the pocket protein familyof cell cycle regulators (i.e. a pocket protein) and an effective amountof an agent that transiently inhibits activity of the cyclin-dependentkinase inhibitor 2A (CDKNA2) alternate reading frame protein (ARF).Contact with these agents occurs under conditions that are sufficient toinduce the PMD cell to transiently become a replication competent cell(RCC) and divide to produce non-tumorigenic progeny that arepost-mitotic immature cells (PMI) of the same lineage as the PMD. Insome embodiments, the PMD dedifferentiates in the course of becoming aRCC. In some embodiments, the PMD is a myocyte, e.g., a cardiomyocyte.In some embodiments, the PMD is a hepatocyte. In some embodiments, thePMD is a neuron, e.g. a dopaminergic neuron. Any tissue that harborspost-mitotic cells (e.g. muscle, brain, skin, pancreas, liver, etc) isembodied herein,

In some embodiments, the pocket protein is retinoblastoma protein (RB).In some embodiments, the agent that transiently inhibits RB activitytransiently inhibits synthesis of RB protein. In some embodiments, theagent that transiently inhibits ARF transiently inhibits synthesis ofARF protein. In some embodiments, about 10% of PMD present in apopulation of cells are induced to become RCC and divide.

In some embodiments, a population of progeny PMI are provided withconditions that promote differentiation, so as to produce a populationof PMD of a desired lineage. In certain embodiments, progeny PMI aretransferred to differentiation conditions by transplanting the progenyto a target site in a subject. In some embodiments, the subject is asubject in need of tissue regeneration therapy, desirably at the targetsite of transplantation.

In some embodiments of the invention, post-mitotic differentiated cells(PMDs) in a tissue are contacted in vivo with an effective amount of anagent that transiently inhibits the activity of a member of the pocketprotein family of cell cycle regulators (i.e. a pocket protein) and aneffective amount of an agent that transiently inhibits activity of thecyclin-dependent kinase inhibitor 2A (CDKNA2) alternate reading frameprotein (ARF), where the contacted PMDs are induced to transientlybecome replication competent cells (RCCs) and divide in situ to producea population of post-mitotic immature cells (PMIs) of the lineage of thetissue. In some embodiments, the PMD dedifferentiates in the course ofbecoming a RCC. In some embodiments, the post-mitotic differentiatedcells are myocytes, e.g. cardiomyocytes. In some embodiments, the PMDare hepatocytes. In some embodiments, the PMD are neurons, e.g.dopaminergic neurons. Any tissue that harbors post-mitotic cells isembodied herein (eg. pancreas),

In some embodiments, the pocket protein is retinoblastoma protein (RB).In some embodiments, the agent that transiently inhibits RB activitytransiently inhibits synthesis of RB protein. In some embodiments, theagent that transiently inhibits ARF transiently inhibits synthesis ofARF protein. In some embodiments, the agent that transiently inhibitsthe activity of the pocket protein and the agent that transientlyinhibits the activity of ARF are administered to a target site in asubject. In some embodiments, the subject is a subject in need of tissueregeneration therapy, desirably at the target site of administration ofthe agent.

In some embodiments, the PMDs are derived from an individual with adisease to characterize that disease (e.g. cortical neurons fromAlzheimer's patients, dopaminergic neurons from Parkinson's patients,cardiomyocytes from patients with heritable and acquired cardiacdiseases, and the like). In some embodiments, the PMDs are derived froman individual that is alive, e.g. the PMDs are from a live tissuebiopsy. In some embodiments, the PMDs are derived from a patient thathas died, i.e. the PMDs are from a cadaver.

In some aspects of the invention, a method is provided for screening acandidate agent for an effect on a disease condition. In these methods,lineage-restricted cells (LRCs) are produced from a post-mitoticdifferentiated cell (PMD) from an individual with a disease condition bymethods described above. The LRCs are transferred to conditions thatpromote differentiation to produce a differentiated population of cells.Cells that are differentiated are contacted with a candidate agent, andthe viability and/or function of the cells in the differentiatedpopulation are compared to the viability and/or function ofdifferentiated cells not contacted with the candidate agent; whereinenhanced viability and/or function of the cells in the differentiatedpopulation contacted with the candidate agent as compared to adifferentiated population not contacted with the candidate agentindicates that the candidate agent will have an effect on the diseasecondition. In some embodiments, the disease condition is a muscledisorder. In some embodiments, the muscle is smooth muscle, skeletalmuscle, or cardiac muscle. In some embodiments, the disease condition isa nervous system disorder. In some embodiments, the nervous systemdisorder is Parkinson's Disease, Alzheimer's Disease, ALS, a disorder ofolfactory neurons, a disorder of spinal cord neurons, or a disorder ofperipheral neurons. In some embodiments, the PMDs are derived from anindividual that is alive. In some embodiments, the PMDs are derived froma cadaver.

In some aspects of the invention, a method is provided for screening acandidate agent for toxicity to a human. In these methods,lineage-restricted cells (LRCs) are produced from a post-mitoticdifferentiated cell (PMD) from a healthy individual by the subjectmethods described above. The LRCs are transferred to conditions thatpromote differentiation to produce a differentiated population of cells.The differentiated population of cells is contacted with a candidateagent, and the viability and/or function of the cells in thedifferentiated population is compared to the viability and/or functionof differentiated cells not contacted with said candidate agent; whereina decrease in viability and/or function of the cells in thedifferentiated population contacted with the candidate agent as comparedto a differentiated population not contacted with the candidate agentindicates that the candidate agent is toxic to a human. In someembodiments, the PMD is a hepatocyte. In some embodiments, the functionof the cells is assess by assessing a cytochrome P450 panel.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Suppression of RB is sufficient for cell-cycle re-entry in C2C12myotubes.

(A) Schematic representation of the treatment of C2C12 myotubes. (B)sqRT-PCR of RB expression timecourse in hours following treatment withmocksi or RBsi. GAPDH expression shown as RNA loading control. (C)Histogram represents BrdU incorporation in myonuclei of myotubes in day4 of differentiation (DM4), at least 36 hrs following RBsi treatment. Aminimum of 500 nuclei were counted from random fields for each trial.(D) Immunofluorescence images from mock treated DM4 C2C12 myotubes and200 nM RBsi-treated myotubes in DM4 and DMS. Myotubes were labeled withprimary antibody for MHC (red) and BrdU (green), as well as with Hoechst33258 dye (blue). Bar, 150 μm. (E) Western blot of protein expressionlevels of RB (100 kDa) in C2C12 myoblasts (GM), myotubes (DM4), andmyotubes at DM3 and DM4, 24 hrs and 48 hrs respectively after treatmentwith RB-siRNA. GAPDH (35 kDa) as a loading control. (F) Histogramrepresenting the levels of RB in GM, in DM4 and in DM4 treated withRB-siRNA for 48 hrs. Samples are normalized to protein levels of DM4myotubes. Growth medium (GM); Myotubes cultured in differentiationmedium for 4 or 5 days (DM4 or DM5 respectively). Error bars indicatethe mean±SE of at least three independent experiments, P value wasdetermined with a nest (*P<0.05, **P<0.01).

FIG. 2. Suppression of RB and p16/19 is necessary for cell-cyclere-entry in primary myotubes. (A) Schematic representation of thetreatment of primary myotubes. (B) Immunofluorescence images from mocksi-Glo treated primary myotubes and RBsi treated myotubes. Myotubes werelabeled with primary antibody for MHC (red) and BrdU (green), as well aswith Hoechst 33258 dye (blue). Bar, 50 μm. (C) Western blot of primarymyotube protein levels in of RB (100 kDa) and p19ARF (20 kDa) in GM andDM5, after mock treatment, TAM treatment or TAM and p16/19si treatment.GAPDH (35 kDa) as loading control. (D) Immunofluorescence imagesindicating BrdU incorporation in TAM and mock si-Glo treated primarymyotubes compared to TAM and p16/19si-RNA treated primary myotubes. (E)sqRT-PCR showing RB and Ink4a (p16/19) expression in primary myotubes aswell as in two different C2C12 myotube populations treated with Mocksior RBsi. (F) sq-PCR amplification using primers for the shared exon 2-3region of the ink4a locus, from genomic DNA prepared from primarymyoblasts and C2C12 myoblasts. (G) Histogram represents BrdUincorporation in primary myotube nuclei at DM5, following suppression ofRB with either siRNA or TAM. (H) Histogram represents BrdU incorporationin primary myotube nuclei following treatment with TAM and siRNAsagainst Ink4a gene products. Growth medium (GM); Myotubes cultured indifferentiation medium for 4 or 5 days (DM4 or DM5 respectively). Aminimum of 500 nuclei were counted from random fields for each trial inF and G. Error bars indicate the mean±SE of at least three independentexperiments. (*P<0.01).

FIG. 3. Decrease in RB and p16/19 levels leads to upregulation ofmitotic machinery. (A) semi-quantitative RT-PCR (sqRT-PCR) analysis ofexpression of Anillin, AuroraB and Survivin in GM, mock-treated DM5, inDM5 with TAM alone or with TAM and p16/19si. (B) Western blot analysisshowing protein levels of AuroraB (38 kDa) and Survivin (20 kDa)following TAM treatment or TAM and p16/19si in DM5. GAPDH (35 kDa) (C)Immunofluorescence images of primary myotubes in DM5 after mocktreatment, and in DM5 after treatment with TAM and p16/19-siRNA.Myotubes were labeled with primary antibody for BrdU (green), Survivin(red), as well as with Hoechst 33258 nuclear dye (blue). Bar, 50 μm. (D)Histogram represents colocalization of BrdU and Survivin in primarymyotube nuclei treated with TAM and non-specific Mock-siRNA duplexes ascompared to myotubes treated TAM and p16/19-siRNA duplexes. Growthmedium (GM); Myotubes cultured in differentiation medium for 4 or 5 days(DM4 or DM5 respectively). A minimum of 500 nuclei were counted fromrandom fields for each trial. Error bars indicate the mean±SE of atleast three independent experiments. (*P<0.005)

FIG. 4. Dedifferentiation of mature myotubes. (A) Immunofluorescenceimages of primary myoblasts and myotubes cultured for indicated times inDM and treated with TAM and either non-specific siRNA (Mocksi) duplexesor p16/19si at the indicated time points. Myotubes labeled with primaryantibodies for MHC (red), BrdU (green), and Hoechst 33258 (blue). Bottompanels phase images of the same time points. Bar, 150 μm. (B) Westernblot analysis of primary myoblasts (GM) and DM5 PM showing expression ofMHC (220 kDa), AuroraB (38 kDa) and Survivin (20 kDa) as well asexpression of these same proteins after myotube treatment with siRNAduplexes against p16/19, with TAM or TAM and p16/19. (C) MHC proteinlevels normalized to differentiated myotube cultures. Primary musclecells were treated with TAM or TAM and p16/19si Growth medium (GM),Differentiation medium (DM). (D) Immunofluorescence images of DM6primary myotubes treated with EtOH and non-specific siRNA duplexes orTAM and p16/19si for at least 48 hrs. Myotubes labeled with primaryantibodies for MHC (red), BrdU (green), and Hoechst 33258 (blue). Growthmedium (GM); Myotubes cultured in differentiation medium for 4 or 5 days(DM4 or DM5 respectively). (E) Immunofluorescence images of DM6 primarymyotubes treated with non-specific siRNA duplexes or TAM and p16/19sifor at least 48 hrs. Myotubes labeled with primary antibodies foralpha-tubulin (green) and Hoechst 33258 nuclear stain (blue). (F, G)Western blot analysis of Myogenin (36 kDa) (F) and alpha tubulin (50kDa) (G) in primary myoblasts (GM) and DM6 (myogenin) on indicated days(tubulin) treated as indicated with siRNA duplexes and/or TAM. In eachof the blots, GAPDH (35 kDa) is the loading control

FIG. 5. Myogenin-expressing myocytes can enter S-phase only after lossof RB and Ink4a genes. (A) Immunofluorescence images of myoblasts (GM)and myocytes (DM3) infected pLE-myog3R-GFP. Cells were labeled withprimary antibodies for GFP (green), myogenin (red) and Hoechst 33258(blue). Bar 50 μm. (B.) (i) Histogram represents percentage ofGFP-positive and myogenin-positive cells in myoblasts (GM) or myocytes(DM3). A minimum of 1000 nuclei were counted from random fields for eachtrial. Error bars indicate the mean±SE of at least three independentexperiments. (*P<0.005). (ii) Histogram represents percentage ofGFP-positive cells that also express myogenin. Individual cells wereevaluated for expression of each marker. A minimum of 250 cells werecounted from random fields. Error bars indicate the mean±SE of threeindependent experiments. (C) Representative FACS plots of myoblasts (GM)and myocytes (DM3) infected with retroviral pLE-myog3R-GFP construct.Gated population indicates GFP-positive myocyte population employed insubsequent experiments. (D) Histogram representation of GPF expressionin three independent experimental FACS profiles on myoblasts (GM) andmyocytes (DM3) (*P<0.001). (E) Immunofluorescence images of GFP-positiveFACS-sorted myocytes cultured in conditioned GM only or in cGM with TAMand p16/19si-RNA. Cells labeled for Ki67 (red) and GFP (green) as wellas Hoechst 33258 (blue). Bar 50 μm. (F) Histogram represents percent ofKi67-positive nuclei in GFP-positive FACS sorted population, in cGM,treated with TAM and p16/19si, or treated with RBsi and p16/19si-RNAduplexes in tandem (DKD). Growth medium (GM). A minimum of 100 nucleiwere counted from random fields for each trial. Error bars indicate themean±SE of three independent experiments. (*P<0.01, **P<0.005).

FIG. 6. (A) (i) Schematic representations of the culture conditions,treatment with mock siRNA, and isolation by laser microdissection andcatapulting of myogenin-GFP+ yocytes. Diagram also shows the fate ofisolated cells 72 and 96 hr after isolation. (ii) Representative imagesof mock-treated myocyte (DM4) (panels left to right) prior tomicrodissection, immediately after microdissection, after LPC isolation,72 hr postisolation, and 96 hr postisolation. Scale bars represent 50 mmand 100 mm. (B) (i) Schematic representations of the culture conditions,treatment with TAM and p16/19 siRNA, and isolation by lasermicrodissection and catapulting of myogenin-GFP+ myocytes. Diagramalsoshows the fate of isolated cells 72 and 96 hr after isolation. (ii)Representative image of TAM- and p16/19siRNA-treated myocyte (DM4):First panel, native GFP expression marks myogenin expression prior tomicrodissection; second panel, the same cell during microdissection;third panel, after LPC isolation; fourth panel, 96 hr postisolation andvisualization of expansion. Scale bars represent 50 mm and 100 mm. (C)(i) Schematic representations of the culture conditions, treatment withRb and p16/19 siRNA, and isolation by laser microdissection andcatapulting of myogenin-GFP+ myocytes. Diagram also shows the fate ofisolated cells 72 and 96 hr after isolation. (ii) Representative imageof DKD-treated myocyte: first panel, GFP expression marks myogeninexpression prior to microdissection; second panel, the same cell aftermicrodissection; third panel, after LPC; fourth panel, 72 hr postisolation with visualization of expansion. Scale bars represent 50 mmand 200 mm. (D) Histogram represents percentage of colony formationafter PALM LPC cell capture as indicated by scheme in FIG. 6A. Errorbars indicate the mean±SE of at least five independent experiments, inwhich at least 50 myocyte membranes were captured for each trial, and atleast 20 myoblast membranes were captured to verify cell captureefficiency. (E) Histogram represents percentage of colony formationafter PALM LPC cell capture following FACS isolation of GFP⁺ myocytepopulation as indicated by the scheme in FIG. 19. Error bars indicatethe mean±SE of at least four independent experiments, in which at least50 myocyte membranes were captured for each trial. Myocytes cultured indifferentiation medium for 4 days (DM4).

FIG. 7. Dedifferentiated myocytes are capable of expansion andredifferentiation into mature myotubes. (A) Phase contrast images of twoDKD captured myocyte colonies and two TAM+p16/19si captured myocytecolonies at DM4, prior to protein harvest for expression analysis. Bar150 μm (B) Western blot analysis of captured colonies in GM and DMarranged from left to right according to their differentiatedmorphologies in DM4; protein levels of RB (100 kDa), p19ARF (20 kDa),myogenin (36 kDa), MHC (220 kDa) and Survivin (20 kDa) as well as GAPDH(35 kDa) as a loading control. (C) Representative images of two DKDcaptured myocyte colonies in DM4, labeled for GFP (green) and myogenin(red) as well as Hoechst 33258 (blue). Bar 25 μm. (D) Representativeimages of TAMcap2 captured myocyte colony in DM4 labeled for MHC (red)and Hoechst 33358 (blue), a subset of which (lower panels) was infectedwith retrovirus re-introducing RB expression. Bar 50 μm. (E) Westernblot analysis of Pax-7 protein (57 kDa) in muscle cells in GM, DM atindicated time points and with indicated treatments, and inproliferating dedifferentiated clones. (F) Western blot analysis ofM-cadherin protein (88 kDa) and MyoD (34 kDa) levels in primary musclecells under growth conditions (GM), differentiated conditions (DM6) withindicated treatments, and in the isolated dedifferentiated clones(DKDcap1 and DKDcap2) in GM and DM4 (DM).

FIG. 8. Dedifferentiated myocytes are capable of fusing to muscle invivo. (A) Representative cross-sections of tibialis anterior 10 dayspost-injection of 2.5×10⁵ cells m TAMcap1 and TAMcap1+RB capturedmyocytes. Incorporation of dedifferentiated myocytes into pre-existingfibers can be visualized in merged fields by GFP⁺ staining (green) of alaminin-bound fibers (red), nuclei (blue); to enhance visualizationcells were infected with constitutive-eGFP expressing retroviral vectorprior to injection. Bar 50 μm. (B) Schematic representation of theevents following suppression of RB and p19ARF in primary differentiatedmyocytes and multi-nucleated myotubes.

FIG. 9. silmporter myotube transfection efficiency. (A) Representativeimages of DM5 C2C12 myotubes transfected with siGlo-Green (non-specificsiRNA dulplex conjugated to alexa-488 molecule) and either Fugene (top)or silmporter (bottom). Myotubes labeled for MHC (red) and Hoechst 33258(blue). Bar 50 μm. (B) Quantification of transfection efficiency ofsiGlo-Green (100 nM) with Fugene, or varying siGlo-Green concentrationswith silmporter. Myotubes cultured in differentiation medium for 5 days(DM5). Error bars indicate the mean±SE of three independent experiments(*P<0.001, **P<0.005).

FIG. 10. Primary myoblast TAM and siRNA treatment analysis. (A) X-galstaining, indicating Cre expression of primary myoblasts in GM treatedwith EtOH or TAM for 24 hrs. Bar 100 μm. (B) X-gal staining, marking Creexpression of primary myotubes in DM5 treated with EtOH or TAM for 24hrs. Bar 200 μm. (C) sqRT-PCR analysis of DM5 primary myotubes after TAMtreatment. TAM was added to DM2 myotube cultures for the indicatedamount time. (D) sqRT-PCR timecourse of RB and p19ARF expression inprimary myotubes after 24 hr TAM treatment. (E) sqRT-PCR analysis of DKDtreatment of primary myotubes with siRNA duplexes for RBsi or p16/19si,delivered in tandem (T), in unison (U) or in unison at half dose-100 nM(hU). Growth medium (GM); Myotubes cultured in differentiation mediumfor 4 or 5 days (DM4 or DM5 respectively). (F) the percentage ofmyotubes that have at least two BrdU positive myonuclei.

FIG. 11. Suppression of p16 alone is insufficient for S-phase re-entryin myotubes. (A) sqRT-PCR analysis of expression levels of p16, p19 andGAPDH, in TAM treated myotubes after treatment with siRNA duplexesagainst p16-only and p16/19. (B) Histogram represents BrdU incorporationin primary myotube nuclei following treatment with TAM and siRNAsagainst p16/19 or p16-only siRNA duplexes. At least 500 nuclei werecounted for each trial. Error bars indicate the mean±SE of twoindependent experiments (*P<0.001)

FIG. 12. Upregulation of mitotic machinery after suppression of Rb andInk4a gene products. (A) Survivin and BrdU co-localization in myoblasts.Immunofluorescence images of primary myoblasts in Growth medium (GM)stained for BrdU (green) Survivin (red) and nuclear dye Hoechst 33258.(B) Immunofluorescence images of primary myotubes treated withnon-specific siRNA duplexes (Mocksi) or TAM and p16/19si. Myotubeslabeled for Eg5 (green), MHC (red) and Hoechst 33258 (blue). (C)sqRT-PCR analysis of expression levels of cyclins D and E1 as well asEmil/FBOX5, in primary myoblasts in GM, DM and in DM treated asindicated.

FIG. 13. Primary myotubes show minimal apoptosis after loss of Rb andp16/19. Representative immunofluorescence images of primary myotubes atday 6 during differentiation treated as indicated and labeled forAnnexinV (green) and propidium iodide (PI).

FIG. 14. Western analysis of protein levels of p53 and p21, in dividingmyoblasts in growth media as well as in differentiation media at day 3(DM3), day 6 (DM6) and DM6 treated as indicated.

FIG. 15. Myotube dedifferentiation following TAM and p16/19 siRNAtreatment. (A) Western analysis of MHC protein (220 kDa) in C2C12myoblasts (GM) and in myotubes (DM) following treatment with siRNAduplexes against Rb. (B) Immunofluorescence images of DM5 C2C12 myotubestreated with non-specific siRNA duplexes and Rbsi for at least 48 hrs.Myotubes labeled with primary antibodies for MHC (red), BrdU (green),and Hoechst 33258 (blue). Bar, 150 μm. (C) sqRT-PCR analysis of theexpression of late differentiation myogenic markers in Growth Medium(GM) or in Differentiation Medium (DM) before and after TAM or TAM andp16/19si treatment. (D) Western blot analysis of M-CK (50 kDa) inprimary myoblasts (GM) and DM5 treated as indicated with siRNA duplexesand/or TAM; the 45-kD band seen in the M-CK blot, is likely due to ashared epitope identified by the antibody. In each of the Westerns,GAPDH (35 kDa) is the loading control.

FIG. 16. Alpha-tubulin levels drop after loss of Rb and p16/19. Upperpanel: Representative phase and immunofluorescence images of primarymyotubes after Mocksi or TAM and p16/19si treatment. Myotubes werelabeled with primary antibodies for alpha-tubulin (green) and Hoechst33258 (blue). Lower panel: Normalized levels of α-Tubulin protein arequantified.

FIG. 17. Myocytes deficient in RB and p16/19 proliferate following FACSisolation. (A) Representative images pLE-myog3R-GFP myocytes FACS sorteddirectly into microwells, imaged before treatment, as well as 72 and 96hrs after DKD treatment. Magnified panel, highlights expansion of onewell following RBsi and p16/19si treatment. Bar 150 μm. (B) Histogramrepresents percentage of GFP-positive cells that also express myogeninfollowing FACS isolation of subset of cells sorted into microwells.Individual cells were evaluated for expression of each marker. A minimumof 250 cells were counted from random fields. Error bars indicate themean±SE of two independent experiments. (C) Histogram represents percentcolony formation observed in microwells at 96 hrs after treatment. Aminimum of 500 wells with at least one cell before treatment counted foreach trial. Error bars indicate the mean±SE of at least threeindependent experiments. (*P<0.005)

FIG. 18. Single-cell isolation and expansion of dedifferentiatedmyogenin-GFP myocytes. (A) Representative images of untreated GFP⁺myocyte (DM4) prior to microdissection, after LPC, and 72 hrspost-isolation. Bar 50 μm. (B) Representative images of TAM+p16/19sitreated GFP⁺ myocytes, prior to microdissection, after LPC, 48 hrspost-isolation, 72 hrs post-isolation, 96 hrs post-isolation, at whichpoint media was changed, and 120 hrs post-isolation. Bar 50 μm, lastpanel bar 200 μm.

FIG. 19. Single-cell isolation and clonal expansion of FACS sortedmyogenin-GFP⁺ myocytes. (A) Schematic representation of myocyte FACSsorting, treatment and PALM LPC isolation. (B) Representative images ofFACS sorted, TAM treated, myocytes (DM4) prior to microdissection, aftermicrodissection, after LPC, and 72 hrs post-isolation. Bar 50 μm. (C)Representative images of FACS sorted myocytes (DM4) treated with TAM+and16/19si prior to microdissection, after microdissection, after LPC, and72 to 73 hrs post-isolation as well as 96 hrs post-isolation. Bar 50 μm,72 hrs panel bar 200 μm.

FIG. 20. PALM isolation, expansion and differentiation of primarymyoblasts and TAM and p16/19si treated myocytes. (A) PALM isolatedmyoblasts at 48 hrs post-isolation, at 96 hrs post-isolation. Threeweeks post-isolation, captured myoblasts were placed in DifferentiationMedium (DM). Images show culture after 3 days in DM (DM3). (B) PALMisolated myogenin-promoter-GFP myocytes treated with TAM and p16/19si,prior to microdissection, 72 hrs post-isolation displaying both phaseand native GFP imaging. Three weeks post-isolation expanded anddedifferentiated myoblasts were placed in DM, and images show cultureafter 3 days in DM (DM3) as well as after 6 days in DM (DM6). Growthmedium (GM).

FIG. 21. Expression patterns of PALM isolated myoblast colonies and DKDtreated myocyte colonies. (A) Western blot analysis of PALM isolatedprimary myoblast colony in GM and DM4, showing protein levels of RB (100kDa), p19ARF (20 kDa), MHC (220 kDa), and Survivin (20 kDa). GAPDH (35kDa) is loading control. (B) sqRT-PCR analysis of PALM isolated primarymyoblasts and DKD treated myocytes in GM and DM4. Growth medium (GM);Myotubes cultured in differentiation medium for 4 or 5 days (DM4 or DM5respectively).

FIG. 22. DKD dedifferentiated, captured and expanded myocytes fuse tomuscle in vivo. Representative fields of cross-sections of tibialisanterior of CB17/SCID mice 10 days post-injection of 1.5×10⁵ cells fromDKD captured and expanded myocytes. Sections were stained for Hoechst33258 (blue), GFP (green) and laminin (orange). Incorporation ofdedifferentiated myocytes into pre-existing fibers can be visualized inmerged fields by GFP⁺ staining of a laminin-bound fibers; GFP expressionmarks myogenin expression in fibers where dedifferentiated myocytesfused. Bar 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Methods for producing cells within a lineage, i.e. lineage-restrictedcells (LRCs), from post-mitotic differentiated cells (PMDs) of the samelineage ex vivo and in vivo are provided, wherein the lineage-restrictedcells may encompass mitotic progenitor cells committed to a cell lineage(MPC), post-mitotic immature cells committed to the cell lineage(post-mitotic immature cell, PMI), and post-mitotic differentiated cellsof the cell lineage (post-mitotic differentiated cell, PMD). The subjectlineage-restricted cells that are produced are useful in tissueregeneration; for drug screening; as experimental models of cellulardifferentiation; for screening in vitro assays to define growth anddifferentiation factors and to characterize genes involved in celldevelopment and regulation; and the like. These cells may be useddirectly for these purposes, or they may be genetically modified toprovide altered capabilities. These and other objects, advantages, andfeatures of the invention will become apparent to those persons skilledin the art upon reading the details of the compositions and methods asmore fully described below.

The term “lineage-restricted cell”, or “LRC”, is used herein to mean acell within a defined lineage. LRCs encompass mitotic progenitor cellscommitted to a defined cell lineage (MPC), post-mitotic immature cellscommitted to a particular type of cell in the cell lineage (post-mitoticimmature cell, PMI), and post-mitotic differentiated cells of that celllineage (post-mitotic differentiated cell, PMD). A lineage-restrictedcell is not pluripotent; in other words, it cannot be induced todifferentiate into all cell types in the embryo. Rather, it isrestricted to differentiating into only the cell or cells of a specifiedlineage. Examples of lineages would be the skeletal muscle lineage, thecardiac muscle lineage, the neuronal lineage, the pancreatic islet celllineage, etc.

The term “mitotic progenitor cell”, or “MPC”, is used to describe amitotic progenitor cell committed to a defined cell lineage. MPC arecells that are able to self-regenerate as well as generate daughtercells of their cell lineage. In other words, an MPC is a mitotic cellthat, upon division, gives rise to a) more MPC and/or b) post-mitoticimmature cells committed to a particular cell type in the cell lineage(post-mitotic immature cells, PMIs). MPCs are recognizable as such bytheir expression of one or more markers, i.e. proteins, RNA, etc. thatare known in the art to be characteristic of an immature state ofdifferentiation for their cell lineage. In addition, progenitor cellsare typically mitotic, and thus incorporate BrdU into their DNA and/orexpress one or more markers, e.g. proteins, that are typically expressedin mitotic cells, e.g. Ki67, PCNA, Anillin, AuroraB, and Survivin. Anexample of an MPC is a progenitor cell of the muscle lineage, namely amyoblast, as it can give rise to more myoblasts and/or post-mitoticmuscle precursors.

The term “post-mitotic immature cell”, or “PMI”, refers to post-mitoticprecursor cells committed to a particular cell type in the cell lineage,that is, post-mitotic cells that have committed to a cell fate but havenot yet differentiated to that cell fate. The PMI typically does nothave all of the defining characteristics of a fully mature cell of thelineage. PMIs of a lineage are recognizable as such by their expressionof one or more markers and a characteristic morphology as is well knownin the art. In addition, PMIs are distinguishable from MPCs because theyare post-mitotic and thus do not incorporate BrdU into their DNA orexpress markers that are typically expressed in mitotic cells, e.g.Ki67, PCNA, Anillin, AuroraB, and Survivin. An example of a PMI is aprecursor cell of the cardiac muscle lineage, as it is post-mitotic buthas not fully differentiated into a cardiomyocyte.

The term “post-mitotic differentiated cell”, or PMD, is used herein torefer to a post-mitotic cell of a cell lineage that has differentiatedinto a mature, functional cell of a tissue. PMDs express markers thatare well-known to the artisan as characteristic of a mature cell fate.In addition, because PMDs are post-mitotic, they do not incorporate BrdUinto their DNA or express markers that are typically expressed inproliferating cells, e.g. Ki67, PCNA, Anillin, AuroraB, Survivin, etc.An example of a PMD is a cardiomyocyte, a myofiber, a hepatocyte, aneuron, and the like.

The term “replication competent cell,” or “RCC”, is used to describe acell that is capable of mitosis. RCC cells may express markerscharacteristic of a MPC or a PMI, i.e. markers that are known in the artto be characteristic of an immature state of differentiation for theircell lineage. Alternatively, they may express markers of a PMD, i.e.markers that are well-known to the artisan as characteristic of a maturecell fate. In either instance, they incorporate BrdU into their DNAand/or express one or more markers, e.g. proteins, that are typicallyexpressed in mitotic cells, e.g. Ki67, PCNA, Anillin, AuroraB, Survivin,and the like.

It will be understood by those of skill in the art that in discussingthe expression of markers by cells as above, the stated expressionlevels reflect detectable amounts of the marker. A cell that is negativefor staining for a marker protein (the level of binding of a markerspecific reagent is not detectably different from an isotype matchedcontrol) may still express minor amounts of the marker. And while it iscommonplace in the art to refer to cells as “positive” or “negative” fora particular marker, actual expression levels are a quantitative trait.For example, the number of molecules on the cell surface can vary byseveral logs, yet still be characterized as “positive”. The stainingintensity of cells can be monitored by flow cytometry, where lasersdetect the quantitative levels of fluorochrome (which is proportional tothe amount of cell surface marker bound by specific reagents, e.g.antibodies). Although the absolute level of staining may differ with aparticular fluorochrome and reagent preparation, the data can benormalized to a control. Alternatively, the staining intensity of cellscan be monitored by immunohistochemistry or immunofluorescence.Cell-specific markers that are cell surface proteins may be observedwithout fixing the cells, i.e. while maintaining cell viability, e.g. byflow cytometry. Alternatively, intracellular markers may be observed ina subpopulation of the subject cells that are fixed and prepared bymethods known in the art.

By “dedifferentiate”, it is meant that cells revert from a moredifferentiated state to a less differentiated state in a cell lineage.In other words, the cells lose traits, e.g. morphology, expression ofcertain genes, functional capabilities etc. of the more differentiatedcell and acquire traits of cells of the lineage that are less mature. By“transiently dedifferentiate,” it is meant that the dedifferentiationphase is temporary; that is, that after a given amount of time and/orunder certain given conditions, the dedifferentiated cells and/or theirprogeny will be permitted to differentiate to a more mature fate, unlikea tumor cell, which is not able to do so.

By “proliferate” it is meant to divide by mitosis, i.e. undergo mitosis.An “expanded population” is a population of cells that has proliferated,i.e. undergone mitosis, such that the expanded population has anincrease in cell number, that is, a greater number of cells, than thepopulation at the outset.

The term “explant” refers to a portion of an organ or tissue thereintaken from the body and cultured in an artificial medium. Cells that aregrown “ex vivo” are cells that are taken from the body in this manner,temporarily cultured in vitro, and returned to the body.

The term “primary culture” denotes a mixed cell population of cells froman organ or tissue within an organ. The word “primary” takes its usualmeaning in the art of tissue culture.

The term “tissue” refers to a group or layer of similarly specializedcells which together perform certain special functions.

The term “organ” refers to two or more adjacent layers of tissue, whichlayers of tissue maintain some form of cell-cell and/or cell-matrixinteraction to form a microarchitecture.

A “pocket protein” is a protein that is a member of the pocket proteinfamily of cell cycle regulators. Pocket proteins are tumor suppressorproteins; in other words, they are negative regulators of the cellcycle. There are three known pocket proteins: Retinoblastoma protein(also called RB, RB1, pRB, OSRC, pp110 or p105-RB), p107 (also calledRBL1, CP107), and p130 (also called RB2). The human RB polypeptidesequence and the nucleotide sequence that encodes it may be found atGenbank Ref. No. NM_(—)000321 (SEQ ID NO:1 and SEQ ID NO:2). The humanp107 polypeptide sequence and the nucleotide sequence that encodes itmay be found at Genbank Ref. Nos. NM_(—)002895 (variant 1; SEQ ID NO:3and SEQ ID NO:4), and NM_(—)183404 (variant 2; SEQ ID NO:5 and SEQ IDNO:6). The human p130 polypeptide sequence and the nucleotide sequencethat encodes it may be found at Genbank Ref. No. NM_(—)005611 (SEQ IDNO:7 and SEQ ID NO:8). These proteins negatively regulate the cell cyclein part by repressing E2F transcription factor activity to limit theexpression of genes required for cell cycle progression. In thecanonical signaling pathway, members of the pocket protein family bindmembers of the E2F family of proteins to prevent E2F-directedtranscription of genes that mediate entry into the cell cycle.Phosphorylation of the pocket proteins or disruption of the pocketprotein-E2F interaction releases the E2F proteins, which can now inducethe transcription of genes that mediate S-phase entry. This and othermechanisms of action of the pocket proteins are described in more detailin Cobrinik (2005) Oncogene 24:2796-2809, the disclosure of which isincorporated herein by reference.

An “agent that transiently inhibits the activity of a pocket protein” isan agent that transiently antagonizes, inhibits or otherwise negativelyregulates the pocket protein's modulation of a cell's activity. Agentsthat transiently inhibit pocket protein activity can act anywhere alongthe pocket protein signaling pathway to antagonize pocket proteinsignaling. Thus, for example, based upon the above described paradigm ofpocket protein signaling, agents that inhibit pocket protein modulationof cell activity include those that prevent the synthesis of the pocketprotein (e.g. siRNAs for RB, p107, or p130), induce the phosphorylationof the pocket protein (e.g. D cyclin peptides); disrupt binding betweenthe pocket protein and E2F (e.g. human papillomavirus peptide E7);overcome the activity of the pocket protein (e.g. E2F peptides); etc.

The “cyclin-dependent kinase inhibitor 2A (CDKNA2) alternate readingframe” (ARF, also known as p14ARF in humans and p19ARF in mice) is thepolypeptide encoded by transcript variant/isoform 4 of thecyclin-dependent kinase inhibitor 2A (CDKNA2, Ink4a, MTS) gene. The ARFpolypeptide sequence and the sequence of the CDKNA2 gene that encodes itmay be found at Genbank Ref. No. NM_(—)058195 (SEQ ID NO:9 and SEQ IDNO:10). The variant is encoded by an alternate first exon located 20 Kbupstream of the remainder of the gene, which renders the ARF proteinstructurally unrelated to the proteins encoded by the other CDKNA2transcript variants p16Ink4a (Genbank Ref. No. NM_(—)000077) and isoform3 (NM_(—)058197, NP_(—)478104). ARF functions as a stabilizer of thetumor suppressor protein p53, in part by interacting with andsequestering HDM2 (MDM2 p53 binding protein homolog, also called HDMXand MDM2), which is normally responsible for the degradation of p53.During mitosis, Not dead yet 1 (NDY1/KDM2b) represses ARF expression byinducing histone H3K27 trimethylation of the ARF locus; in the absenceof ARF protein, HDM2 proteins are available to degrade p53, therebyrelieving p53-mediated suppression of mitosis. This and other mechanismsof action of the ARF protein are well known in the art.

An “agent that transiently inhibits the activity of ARE” is an agentthat transiently antagonizes, inhibits or otherwise negatively regulatesthe ARF modulation of cell activity. Agents that inhibit ARF activitycan act anywhere along the ARF signaling pathway to antagonize ARFsignaling. Thus, for example, based upon the above described paradigm ofARF signaling, agents that inhibit ARF modulation of cell activityinclude those that inhibit the expression of ARF (e.g. NDY1 peptide),prevent the synthesis of the ARF protein (e.g. ARF siRNA), overcome theactivity of the ARF protein (e.g. HDM2 peptide), etc.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

As summarized above, the subject invention provides methods forproducing cells within a lineage, i.e. lineage-restricted cells (LRCs),from post-mitotic differentiated cells (PMDs) of the same lineage exvivo and in vivo, wherein the lineage-restricted cells may encompassmitotic progenitor cells committed to a cell lineage (mitotic progenitorcells, MPC), post-mitotic immature cells committed to a particular typeof cell in the cell lineage (post-mitotic immature cell, PMI), andpost-mitotic differentiated cells of that lineage (post-mitoticdifferentiated cell, PMD). In further describing the subject invention,the subject methods are described first in greater detail, followed by areview of various representative applications in which the subjectinvention finds use as well as kits that find use in practicing thesubject invention.

In practicing the subject methods, post-mitotic differentiated cells(PMDs) are contacted with an agent that transiently inhibits activity ofone or more members of the pocket protein family of cell cycleregulators, and an agent that transiently inhibits activity of thetranscript variant of the cyclin-dependent kinase inhibitor 2A referredto as ARF. As defined above, PMDs are cells that have completeddifferentiation to become mature, functional cells in a tissue, e.g. amyocyte in skeletal or heart muscle, an islet cell in pancreas, ahepatocyte in liver, a neuron in CNS tissue or peripheral neural tissue,an osteocyte in bone, hematopoietic cell from blood, etc. PMDs can beidentified as such by the expression of one or more proteins or RNAs,i.e. markers, as will be known in the art. In addition, these cells mayexpress one or more subtype-specific markers, as will also be known inthe art. In some embodiments, the subject PMDs cells are myocytes, whichexpress one or more of myogenin, myosin heavy chain (MHC), and creatinekinase. In certain embodiments, the myocytes are cardiomyocytes, whichare rod shaped and cross-striated in culture and express one or more ofproteins cardiac troponin, eHand transcription factor, andcardiac-specific myosins. In certain embodiments, the myocytes aresmooth muscle myocytes, which express smooth muscle actin. In certainembodiments, the myocytes are skeletal muscle myocytes, which expressone or more of skeletal muscle myosins, skeletal muscle troponin, myoD.

In some embodiments, subject PMDs are contacted with agents in vivo,that is, in the tissue in which they reside, i.e. in situ. In someembodiments, subject PMDs are contacted with agents ex vivo, that is,they are harvested from the body and contacted with agents in vitro. Incases when the method is to be performed ex vivo, the PMDs may becultured from an explant, e.g. biopsy or autopsy material, as a cultureof primary cells. Methods of culturing PMDs from explants are typicallyspecific for the type of primary cell being cultured, and are well knownto one of ordinary skill in the art. As one non-limiting example, forembodiments wherein the PMDs are myocytes, exemplary methods may befound in Mitcheson, J S et al. (1998) Cardiovascular Research39(2):280-300 (for cardiomyocytes); Rosenblatt et al. (1995) In VitroCell Dev. Biol Anim 31(10):773-339 (for human skeletal muscle myocytes);Siow, R C M and Pearson, J D (2001) Methods in Molecular Medicine:Angiogenesis protocols 46:237-245 (vascular smooth muscle myocytes); andGraham M, and Willey A. (2003) Methods in Molecular Medicine: Woundhealing 78:417-423 (intestinal smooth muscle myocytes), the disclosuresof which are incorporated herein by reference.

Subject PMDs are contacted ex vivo or in vivo with an effective amountof an agent that transiently inhibits RB activity and an effectiveamount of an agent that transiently inhibits ARF activity. As discussedabove, an agent that transiently inhibits activity of a pocket proteinfamily member is an agent that transiently antagonizes, inhibits orotherwise negatively regulates the pocket protein's modulation of acell's activity; agents that transiently inhibit pocket protein activitycan therefore act anywhere along a pocket protein signaling pathway asit is known in the art and described above to antagonize pocket proteinsignaling. Similarly, an agent that transiently inhibits the activity ofARF is an agent that transiently antagonizes, inhibits or otherwisenegatively regulates the ARF modulation of cell activity; agents thatinhibit pocket protein activity can therefore act anywhere along thepocket protein signaling pathway as it is known in the art and describedabove to antagonize pocket protein signaling By an effective amount ofagent, it is an amount that will transiently reduce the overall activityof the subject pathway by at least about 25%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, by about100%, such that the cell is now able to enter mitosis and divide. Inother words, the overall activity of the subject pathway will be reducedby at least about 2-fold, usually by at least about 5-fold, e.g.,10-fold, 15-fold, 20-fold, 50-fold, 100-fold or more, as compared to acontrol ,i.e. uncontacted, cell. For agents that transiently inhibit theactivity of a pocket protein, this biochemically may be realized by, forexample, reducing the amount of pocket protein in the cell by about 10%or 50% or more, 70% or more, 90% or more, or up to 100%; increasing theamount of phosphorylation of the pocket protein by about 10% or 50% ormore, by 70% or more, by 100% or more, by 200% or more, by 500% or more;increasing the amount of free E2F in the cell by about 10% or more, 25%or more, 50% or more, 100% or more, 200% or more, 500% or more; etc. asis well known in the art. For agents that transiently inhibit theactivity of ARF, this biochemically may be realized by, for example,increasing the amount of free NDY1 in the cell by about 10% or more, 25%or more, 50% or more, 100% or more, 200% or more, 500% or more; reducingthe amount of ARF in the cell by about 10% or 30% or more, 50% or more,70% or more, about 100%; increasing the amount of free HDM2 in the cellby about 10% or more, 25% or more, 50% or more, 100% or more, 200% ormore, 500% or more; etc. as is well known in the art. By transiently, itis meant that the inhibition is for a limited period of time. Intransiently inhibiting a target pathway, an effective amount of agentwill antagonize its target pathway for about 12 hours, about 1 day,about 2 days, about 3 days, about 5 days, about 7 days, about 10 days,about 15 days, about 20 days, or about 30 days. Antagonism of the targetpathway may cease either naturally, e.g. because the agent is degraded,naturally inactivated over time, removed from the body of a subject bythe blood, etc. or it may be actively shut off, e.g. by providingadditional agents that inhibit expression or activity of the subjectagent.

Agents suitable for transiently inhibiting pocket protein activity andARF activity in the present invention include small molecule compounds.Naturally occurring or synthetic small molecule compounds of interestinclude numerous chemical classes, such as organic molecules, e.g.,small organic compounds having a molecular weight of more than 50 andless than about 2,500 daltons. Candidate agents comprise functionalgroups for structural interaction with proteins, particularly hydrogenbonding, and typically include at least an amine, carbonyl, hydroxyl orcarboxyl group, preferably at least two of the functional chemicalgroups. The candidate agents may include cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Exemplary of pharmaceutical agents suitable forthis invention are those described in “The Pharmacological Basis ofTherapeutics” (Goodman and Gilman (1996) McGraw-Hill, New York, N.Y.,Ninth edition). Also included are toxins, and biological and chemicalwarfare agents, for example see Somani, S. M. (Ed.), “Chemical WarfareAgents,” Academic Press, New York, 1992. Small molecule compounds can beprovided directly to the medium in which the cells are being cultured,for example as a solution in DMSO or other solvent.

Agents suitable for transiently inhibiting pocket protein activity andARF activity in the present invention also include nucleic acidmolecules that inhibit the synthesis of pocket proteins, ARF proteins,and/or other proteins of the pocket protein or ARF pathways,respectively, for example, nucleic acids that encode antisense, siRNA,or shRNA molecules that target the RB, p107, p130 or CDKNA2/Ink4a genesand transcripts.

For example, nucleic acid agents of interest include antisenseoligonucleotides (ODN), particularly synthetic ODN having chemicalmodifications from native nucleic acids, or nucleic acid constructs thatexpress such antisense molecules as RNA. The antisense sequence iscomplementary to the targeted coding sequence, and inhibits itsexpression. Antisense molecules may be produced by expression of all ora part of the target coding sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisenseoligonucleotide is a synthetic oligonucleotide. Antisenseoligonucleotides will generally be at least about 7, usually at leastabout 12, more usually at least about 20 nucleotides in length, and notmore than about 25, usually not more than about 23-22 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like.Antisense oligonucleotides may be chemically synthesized by methodsknown in the art. Preferred oligonucleotides are chemically modifiedfrom the native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications that alter the chemistry of the backbone, sugars orheterocyclic bases have been described in the literature. Among usefulchanges in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The alpha-anomer of deoxyribose may be used, where the base isinverted with respect to the natural beta-anomer. The 2′-OH of theribose sugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars,which provides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively. One or a combination of antisenseoligonucleotides may be administered, where a combination may comprisemultiple different sequences.

As another example, nucleic acid agents of interest include RNA agentsthat inhibit the synthesis of pocket proteins, ARF protein, or proteinsin the pathways activated by these subject proteins. By RNA agents it ismeant ribonucleotide-based agents that modulate expression of targetgenes, i.e. RB, p107, p130, or ARF, or members of their signalingpathways, by an RNA interference mechanism. The RNA agent may be amicroRNA; see, e.g. Mudhasani et al. (2008) J Cell Biol 181(7):1055-63,which teaches that miRNAs suppress the expression of ARF. The RNA agentmay be an RNAi agent, e.g., a small ribonucleic acid molecule (alsoreferred to herein as an interfering ribonucleic acid), i.e.,oligoribonucleotides, that is present in duplex structures, e.g., twodistinct oligoribonucleotides hybridized to each other (siRNA) or asingle ribooligonucleotide that assumes a small hairpin formation toproduce a duplex structure (shRNA). By oligoribonucleotide is meant aribonucleic acid that does not exceed about 100 nt in length, andtypically does not exceed about 75 nt length, where the length incertain embodiments is less than about 70 nt. Where the RNA agent is aduplex structure of two distinct ribonucleic acids hybridized to eachother, i.e. an siRNA, the length of the duplex structure typicallyranges from about 15 to 30 bp, usually from about 15 to 29 bp, wherelengths between about 20 and 29 bps, e.g., 21 bp, 22 bp, are ofparticular interest in certain embodiments. Where the RNA agent is aduplex structure of a single ribonucleic acid that is present in ahairpin formation, i.e., a shRNA, the length of the hybridized portionof the hairpin is typically the same as that provided above for thesiRNA type of agent or longer by 4-8 nucleotides. The weight of the RNAiagents of this embodiment typically ranges from about 5,000 daltons toabout 35,000 daltons, and in many embodiments is at least about 10,000daltons and less than about 27,500 daltons, often less than about 25,000daltons.

dsRNA can be prepared according to any of a number of methods that areknown in the art, including in vitro and in vivo methods, as well as bysynthetic chemistry approaches. Examples of such methods include, butare not limited to, the methods described by Sadher et al. ((1987)Biochem. Int. 14:1015); by Bhattacharyya ((1990) Nature 343:48); byLivache, et al. (U.S. Pat. No. 5,795,715); by Sambrook, et al. ((1989)Molecular Cloning: A Laboratory Manual, 2nd ed.); in Transcription andTranslation ((1984) B. D. Hames, and S. J. Higgins, Eds.); in DNACloning, volumes I and II ((1985) D. N. Glover, Ed.); and inOligonucleotide Synthesis ((1984) M. J. Gait, Ed.), each of which isincorporated herein by reference in its entirety. Single-stranded RNA(ssRNA) can also be produced using a combination of enzymatic andorganic synthesis or by total organic synthesis. The use of syntheticchemical methods enable one to introduce desired modified nucleotides ornucleotide analogs into the dsRNA or ssRNA. For example, smallinterfering double-stranded RNAs (siRNAs) engineered with certain‘drug-like’ properties such as chemical modifications for stability andcholesterol conjugation for delivery have been shown to achievetherapeutic silencing of an endogenous gene in vivo. To develop apharmacological approach for silencing target genes in vivo, chemicallymodified, cholesterol-conjugated single-stranded RNA analoguescomplementary to sequence in target genes may be synthesized usingstandard solid phase oligonucleotide synthesis protocols. The RNAs areconjugated to cholesterol, and may further have a phosphorothioatebackbone at one or more positions. RNA inhibitory agents may be providedat a final concentration of about 50 nM-500 nM, more usually 100 nM-200nM.

In certain embodiments, the RNA agent may be a transcriptional templateof the interfering ribonucleic acid. In these embodiments, thetranscriptional template is typically a DNA that encodes the interferingribonucleic acid. The DNA may be present in a vector, where a variety ofdifferent vectors are known in the art, e.g., a plasmid vector, a viralvector, etc.

Agents suitable for transiently inhibiting the pocket protein activityand ARF activity in the present invention also include polypeptides,e.g. dominant negative peptides, or peptides of targets of the pocketproteins or ARF that are normally antagonized by pocket protein or ARFactivity as is understood in the art. Such polypeptides may optionallybe fused to a polypeptide domain that increases solubility of theproduct. The domain may be linked to the polypeptide through a definedprotease cleavage site, e.g. a TEV sequence, which is cleaved by TEVprotease. The linker may also include one or more flexible sequences,e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage ofthe fusion protein is performed in a buffer that maintains solubility ofthe product, e.g. in the presence of from 0.5 to 2 M urea, in thepresence of polypeptides and/or polynucleotides that increasesolubility, and the like. Domains of interest include endosomolyticdomains, e.g. influenza HA domain; and other polypeptides that aid inproduction, e.g. IF2 domain, GST domain, GRPE domain, and the like.

To promote delivery of the polypeptide to the intracellular domain ofthe cell, the polypeptide may comprise the polypeptide sequences ofinterest fused to a polypeptide permeant domain. A number of permeantdomains are known in the art and may be used in the non-integratingpolypeptides of the present invention, including peptides,peptidomimetics, and non-peptide carriers. For example, a permeantpeptide may be derived from the third alpha helix of Drosophilamelanogaster transcription factor Antennapaedia, referred to aspenetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK. Asanother example, the permeant peptide comprises the HIV-1 tat basicregion amino acid sequence, which may include, for example, amino acids49-57 of naturally-occurring tat protein. Other permeant domains includepoly-arginine motifs, for example, the region of amino acids 34-56 ofHIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, forexample, Futaki et al. (2003) Curr Protein Pept Sci. April 2003; 4(2):87-96; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A Nov. 21,2000; 97(24):13003-8; published U.S. Patent applications 20030220334;20030083256; 20030032593; and 20030022831, herein specificallyincorporated by reference for the teachings of translocation peptidesand peptoids). The nona-arginine (R9) sequence is one of the moreefficient PTDs that have been characterized (Wender et al. 2000; Uemuraet al. 2002).

Agents suitable for transiently inhibiting pocket protein activity andARF activity in the present invention also include nucleic acids thatencode the aforementioned polypeptides that antagonize pocket proteinactivity and ARF activity. Many vectors useful for transferring nucleicacids into target cells are available. The vectors may be maintainedepisomally, e.g. as plasmids, minicircle DNAs, virus-derived vectorssuch cytomegalovirus, adenovirus, etc., or they may be integrated intothe target cell genome, through homologous recombination or randomintegration, e.g. retrovirus derived vectors such as MMLV, HIV-1, ALV,etc. Vectors may be provided directly to the subject cells. In otherwords, the subject post-mitotic differentiated cells are contacted withvectors comprising the nucleic acid of interest such that the vectorsare taken up by the cells. Methods for contacting cells with nucleicacid vectors, such as electroporation, calcium chloride transfection,and lipofection, are well known in the art.

Alternatively, the nucleic acid of interest may be provided to thesubject cells via a virus. In other words, the subject post-mitoticdifferentiated cells are contacted with viral particles comprising thenucleic acid of interest. Retroviruses, for example, lentiviruses, areparticularly suitable to the method of the invention. Commonly usedretroviral vectors are “defective”, i.e. unable to produce viralproteins required for productive infection. Rather, replication of thevector requires growth in a packaging cell line. To generate viralparticles comprising nucleic acids of interest, the retroviral nucleicacids comprising the nucleic acid are packaged into viral capsids by apackaging cell line. Different packaging cell lines provide a differentenvelope protein to be incorporated into the capsid, this envelopeprotein determining the specificity of the viral particle for the cells.Envelope proteins are of at least three types, ecotropic, amphotropicand xenotropic. Retroviruses packaged with ecotropic envelope protein,e.g. MMLV, are capable of infecting most murine and rat cell types, andare generated by using ecotropic packaging cell lines such as BOSC23(Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearingamphotropic envelope protein, e.g. 4070A (Danos et al, supra.), arecapable of infecting most mammalian cell types, including human, dog andmouse, and are generated by using amphotropic packaging cell lines suchas PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Milleret al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988)PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelopeprotein, e.g. AKR env, are capable of infecting most mammalian celltypes, except murine cells. The appropriate packaging cell line may beused to ensure that the subject CD33+ differentiated somatic cells aretargeted by the packaged viral particles. Methods of introducing theretroviral vectors comprising the nucleic acid encoding thereprogramming factors into packaging cell lines and of collecting theviral particles that are generated by the packaging lines are well knownin the art.

Vectors used for providing nucleic acid of interest to the subject cellswill typically comprise suitable promoters for driving the transientexpression, that is, transient transcriptional activation, of thenucleic acid of interest. These will typically be inducible promoters,such as promoters that respond to the presence of drugs such astetracycline. By transcriptional activation, it is intended thattranscription will be increased above basal levels in the target cell byat least about 10 fold, by at least about 100 fold, more usually by atleast about 1000 fold.

One or more agents that transiently inhibit the activity of one or morepocket proteins may be used. Likewise, one or more agents thattransiently inhibit the activity of ARF may be used. The agent(s) may beprovided to the subject post-mitotic differentiated cells individuallyor as a single composition, that is, as a premixed composition, ofagents. When provided individually, the agents may be added to thesubject post-mitotic differentiated cells simultaneously or sequentiallyat different times. For example, the agent(s) that transiently inhibitsthe activity of the pocket protein(s) may be provided first, and theagent(s) that transiently inhibits the activity of ARF is providedsecond, e.g. 24 hours later.

In some embodiments, additional agents that promote mitosis may beprovided to the cell at the contacting step, e.g. growth factors, e.g.bFGF, EGF, BMP, neuregulin, periostin, etc. In some embodiments, agentsthat promote cell cycle reentry are also provided to the cell in thecontacting step. For example, in embodiments in which the subjectpost-mitotic differentiated cell is a skeletal muscle myocyte, agentsthat disrupt microtubules such a myoseverin peptide (Rosania G R et al.(2000) Nat Biotechnol. 18(3):304-8) or a small molecule as is known inthe art (see, e.g., Duckmanton A, (2005) Chem Biol. 12(10):1117-26, thedisclosure of which is incorporated herein by reference) may be providedto fragment the multinucleated skeletal muscle cell. Such agents aretypically used when the subject post-mitotic differentiated cell has amorphologically complex phenotype, for example, a cytoskeletalarchitecture that polarizes the cell, such as the architecture of amultinucleated muscle cell, neuron, hepatocyte, etc.

In embodiments in which the PMDs are induced to become RCCs and dividein vivo, i.e. in situ, agents are administered locally, that is,directly to the target site in a subject, i.e. the tissue. The agentsmay be provided in any number of ways that are known in the art, e.g. asa liquid (e.g. in any suitable buffer (saline, PBS, DMEM, Iscove'smedia, etc.)), as a paste, in a matrix support, conjugated to a solidsupport (e.g. a bead, a filter such as a mesh filter, a membrane, athread, etc), etc. The conditions in the tissue are typically permissiveof dedifferentiation and division of PMDs, and no alteration of thebasal conditions is required with the exception of providing the agentsas described above.

In embodiments in which the PMDs are induced to become RCCs and divideex vivo, the cells are contacted with the agents for about 30 minutes toabout 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6hours, 8 hours, 12 hours, 18 hours, 20 hours, or any other period fromabout 30 minutes to about 24 hours in a culture media that is typicallyused to promote proliferation of progenitor cells of the subject lineageas is known in the art. For example, in embodiments in which thepost-mitotic differentiated cell is a myocyte, the agents may beprovided in DMEM LG+F10 media that has been supplemented with highlevels of serum, e.g. 15%-20% FBS, 1% Pen-Strep, and 1.25-2.5 ng/mLbFGF. In some embodiments, the cells are contacted with agentrepeatedly, e.g. with a frequency of about every day to about every 4days, e.g., every 1.5 days, every 2 days, every 3 days, every 4 days, orany other frequency from about every day to about every four days withthe agent. For example, agents may be provided to the subject cellsonce, and the cells allowed to incubate with the agent for 16-24 hours,after which time the media is replaced with fresh media and the cellsare cultured further, or the agents may be provided to the subject cellstwice, with two 16-24 hour incubations with the agent following eachprovision, after which the media is replaced with fresh media and thecells are cultured further.

Transient inhibition of one or more pocket proteins and ARF willtransiently induce the subject PMDs to become RCCs and divide. Bytransiently induced, it is meant that the PMDs are induced to undergomitosis for a limited amount of time, i.e. 12 hours, 1 day, 2 days, 3days, 5 days, or 7 days. Accordingly, the subject PMDs and their progenymay undergo 1 round of mitosis, up to 2 rounds of mitosis, up to 3rounds, up to 4 rounds, up to 5 rounds, up to 6 rounds, or up to 10rounds of mitosis. This is unlike tumorigenic cells, which undergounregulated mitosis, i.e., continue to divide for an unlimited amount oftime. The period of time in which the RCCs are actively dividing isknown as the induction period. During the induction period, a PMD thatis transiently induced to divide will give rise to a population, orcohort, of progeny that are lineage-restricted cells. In other words, aPMD may give rise to 2 or more cells, 4 or more cells, 8 or more cells,16 or more cells, 32 or more cells, 64 or more cells, 100 or more cells,1000 or more cells, or 10,000 or more cells. In some embodiments,multiple PMDs, i.e. a population of PMDs, are induced to divide, givingrise to a population of progeny that are lineage-restricted cells. Atleast about 1%, about 2%, about 5%, about 8%, more usually about 10%,about 15%, about 20%, or about 50% of contacted post-mitoticdifferentiated cells in a population may be induced to divide. In someembodiments, the PMD dedifferentiates in the course of becoming an RCC.In some instances, transient induction may be controlled by providingagents, e.g. Rb and/o ARF-related agents to return to cells to apost-mitotic state. Examples of such RB and/or or ARF related-agentsinclude RB or ARF polypeptides or the cDNA encoding these polypeptidesor the active domains thereof.

The progeny of these divisions are all cells of a particular lineage,i.e. lineage-restricted cells (LRCs), that lineage being the lineage ofthe PMD that was contacted at the outset. While the agents that inhibitthe pocket protein and ARF are active, i.e. during the induction period,the LRC of the culture may be mitotic progenitor cells (MPCs) that arecommitted to the cell lineage of the PMD. Once the agents that inhibitthe pocket protein and ARF are no longer active, i.e. following thecompletion of the induction period, the LRC of the culture may bepost-mitotic immature cells (PMIs) that are committed to the celllineage of the PMD. For example, in embodiments in which thepost-mitotic differentiated cell(s) is a myocyte, the progeny LRC duringthe induction period may be myoblasts (identifiable for their expressionof one of more mitotic markers as well as one or more myoblast markerssuch as myf5 and pax7) and the progeny LRC after the induction periodmay be muscle precursors (identifiable for their lack of expression ofmitotic markers as well as expression of one of more markers includingmyogenin).

In embodiments in which the PMDs are induced to become RCCs and dividein vivo, i.e. in situ, progeny may spontaneously differentiate into PMDof the lineage, or they may be provided with agents that promotedifferentiation into PMD of that lineage. Likewise, in embodiments inwhich the PMDs are induced to become RCCs and divide ex vivo, theprogeny may spontaneously differentiate into PMD of the lineage, or theymay be transferred to conditions that promote differentiation into themature population of post-mitotic differentiated cells that they aredestined to become. In certain embodiments, the transferring of thecells induced to divide ex vivo to condition that promotedifferentiation is effected by transplanting the progeny into the tissueof a subject. Cells may be transplanted by any of a number of standardmethods in the art for delivering cells to tissue, e.g. injecting themas a suspension in a suitable buffer (saline, PBS, DMEM, Iscove's media,etc.), providing them on a solid support, e.g. a bead, a filter such asa mesh filter, a membrane, etc. In certain embodiments, the transferringis effected by changing the culture media to a media that promotes thedifferentiation of cells of that lineage, as is known in the art. Forexample, in embodiments in which the post-mitotic differentiated cell isa myocyte, the LRC that is produced may be induced to differentiate inDMEM LG media that has been supplemented with low levels of serum, e.g.2% HS, 1% Pen-Strep.

In Vitro Uses

LRCs produced by the subject methods find many uses, both in vitro andin vivo. For example, PMDs derived from individuals having a diseasehave the desired cell-specific identity and disease-related aberrantregulatory program. LRCs produced from these PMDs by the subject methodswill also exhibit the disease phenotype. Thus, the propagated LRCs mayserve as material for the characterization of the regulatory mechanismsthat have gone awry. In addition, these cells will serve as material onwhich to screen therapeutic agents for their ability to ameliorate thedisease phenotype. Accordingly, LRCs produced ex vivo may be used tostudy the regulatory networks or underlying mechanisms that lead to thedisease phenotype. Likewise, LRCs from such diseases may be used toscreen candidate therapeutic agents for efficacy and/or for toxicity inameliorating the regulatory step(s) that have gone awry in the diseasestate.

In screening assays for biologically active agents, LRCs are producedfrom PMDs from an individual, e.g. an individual with a diseasecondition, e.g. a live individual or a cadaver, by the subject methodsdescribed above, and allowed to differentiate. The differentiated cellsare then contacted with a candidate agent of interest and the effect ofthe candidate agent is assessed by monitoring one or more outputparameters. These output parameters may be reflective of an apoptoticstate, such as the amount of DNA fragmentation, the amount of cellblebbing, the amount of phosphatidylserine on the cell surface asvisualized by Annexin V staining, and the like by methods describedabove. Alternatively or additionally, the output parameters may bereflective of the viability of the culture, e.g. the number of cells inthe culture, the rate of proliferation of the culture. Alternatively oradditionally, the output parameters may be reflective of the health ofthe cells in the culture, e.g. the length of time that the cells survivein the culture, the presence or absence of ubiquitin-related puncat inthe culture, etc. Alternatively or additionally, the output parametersmay be reflective of the function of the cells in the culture, e.g. thecytokines and chemokines produced by the cells, the rate of chemotaxisof the cells, the cytotoxic activity of the cells, etc. Such parametersare well known to one of ordinary skill in the art and.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

PMDs useful for producing LRCs include any post-mitotic cell from anytissue comprising post-mitotic cells, e.g. muscle, nervous system,pancreas, liver, etc., e.g. a cardiomyocyte from an individual with aheart condition, a neuron from an individual with Alzheimer's disease,Parkinson's Disease, ALS, etc., as described above.

Candidate agents of interest for screening include known and unknowncompounds that encompass numerous chemical classes, primarily organicmolecules, which may include organometallic molecules, inorganicmolecules, genetic sequences, etc. An important aspect of the inventionis to evaluate candidate drugs, including toxicity testing; and thelike.

Candidate agents include organic molecules comprising functional groupsnecessary for structural interactions, particularly hydrogen bonding,and typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, frequently at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules, including peptides, polynucleotides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Included arepharmacologically active drugs, genetically active molecules, etc.Compounds of interest include chemotherapeutic agents, hormones orhormone antagonists, etc. Exemplary of pharmaceutical agents suitablefor this invention are those described in, “The Pharmacological Basis ofTherapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996),Ninth edition. Also included are toxins, and biological and chemicalwarfare agents, for example see Somani, S. M. (Ed.), “Chemical WarfareAgents,” Academic Press, New York, 1992).

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Candidate agents are screened for biological activity by adding theagent to at least one and usually a plurality of cell samples, usuallyin conjunction with cells lacking the agent. The change in parameters inresponse to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype. In some embodiments, the cells arealso contacted with an agent that suppresses DSBR, further sensitizingthe cells to the apoptotic effects of the elevated numbers of DSBs.

Various methods can be utilized for quantifying the selected parameters.For example, terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) may be employed to measure DNA fragmentation. Flowcytometry may be employed to detect Annexin V binding tophosphatidylserine on the cell surface. BrdU labeling may be employed todetect proliferation rates. Western blots may be employed to assaycytokines and chemokines secreted into the medium. Migration assays,e.g. in Boyden chambers, may be employed to assay chemotaxis capacity.Antibody-dependent cell-mediated cytotoxicity (ADCC) assays may beemployed to assay cytotoxicity of cells. Such methods would be wellknown to one of ordinary skill in the art.

As another example, LRCs produced ex vivo by the subject methods may beused in research, e.g to elucidate cellular mechanisms of disease. Forexample, LRCs produced ex vivo may be characterized to determine themechanisms underlying the disease state and the regulatory pathways thathave gone awry. As another example, the genomic DNA and/or RNA of LRCsproduced ex vivo may be harvested and profiled to better understandwhich promoters are more active and which genes are more highlytranscribed in which cell lineages and at which stages in development.

LRCs produced ex vivo by the subject methods may also be used toidentify novel targets for drugs leading to drug discovery. Agents canbe screened for those that modulate particular signaling pathways tobetter understand the roles that these signaling pathways play in thedifferentiation of cells of a particular lineage. Such agents mayconstitute new therapeutics that ameliorate the disease state.

Additionally, LRCs produced by the subject methods may be used to screenfor compounds for their toxicity. For example, LRCs may be prepared fromhepatocytes using the subject methods, and those LRCs differentiatedinto hepatocytes by the subject methods. The newly differentiated LRCsmay then be used to screen drugs for toxicity. Examples of parametersindicative of cell function that may be assayed in such screens includemembers of the cytochrome P450 panel, or “CYP”, as well known in theart.

In Vivo Uses

As another example, LRCs have the potential to contribute to the tissuefrom which the starting PMDs were acquired, and thus LRCs produced exvivo or in vivo may be used for reconstituting or supplementingdifferentiating or differentiated cells in a recipient, that is, forregenerating tissue. For example, in embodiments of the above methods inwhich the subject post-mitotic differentiated cells are myocytes,transplanting lineage-restricted cells generated by ex vivo methodsdescribed above into muscle, or producing lineage-specific cells in situin the muscle by in vivo methods described above results in thedifferentiation of new muscle cells in the patient. Muscle regenerationas used herein refers to the process by which new muscle fibers formfrom muscle progenitor cells or muscle precursor cells. A therapeuticcomposition will usually confer an increase in the number of new fibersby at least 1%, more preferably by at least 20%, and most preferably byat least 50%. The growth of muscle may occur by the increase in thefiber size and/or by increasing the number of fibers. The growth ofmuscle may be measured by an increase in wet weight, an increase inprotein content, an increase in the number of muscle fibers, an increasein muscle fiber diameter; etc. An increase in growth of a muscle fibercan be defined as an increase in the diameter where the diameter isdefined as the minor axis of ellipsis of the cross section.

Muscle regeneration may also be monitored by the mitotic index ofmuscle. For example, cells may be exposed to a labeling agent for a timeequivalent to two doubling times. The mitotic index is the fraction ofcells in the culture which have labeled nuclei when grown in thepresence of a tracer which only incorporates during S phase (i.e., BrdU)and the doubling time is defined as the average S time required for thenumber of cells in the culture to increase by a factor of two.Productive muscle regeneration may be also monitored by an increase inmuscle strength and agility.

Muscle regeneration may also be measured by quantitation of myogenesis,i.e.

fusion of myoblasts to yield myotubes. An effect on myogenesis resultsin an increase in the fusion of myoblasts and the enablement of themuscle differentiation program. For example, the myogenesis may bemeasured by the fraction of nuclei present in multinucleated cells inrelative to the total number of nuclei present. Myogenesis may also bedetermined by assaying the number of nuclei per area in myotubes or bymeasurement of the levels of muscle specific protein by Westernanalysis.

The survival of muscle fibers may refer to the prevention of loss ofmuscle fibers as evidenced by necrosis or apoptosis or the prevention ofother mechanisms of muscle fiber loss. Muscles can be lost from injury,atrophy, and the like, where atrophy of muscle refers to a significantloss in muscle fiber girth.

Tissue regeneration therapy that employs the lineage-restricted cellsproduced by the subject methods are useful for treating subjectssuffering from a wide range of diseases or disorders. For example, inembodiments in which the post-mitotic differentiated cells are myocytes,subjects suffering from muscular disorders, e.g., acute cardiacischemia, injury due to surgery (e.g. tumor resection) or physicaltrauma (amputation/gunshot wound), or degenerative heart diseases suchas ischemic cardiomyopathy, conduction disease, and congenital defects,etc. could especially benefit from regenerative tissue therapies thatuse the lineage-restricted cells of the subject method.

Particular examples of muscle disorders that could be treated with thesubject cells include disorders of the heart muscle. Such disordersinclude, without limitation, myocardial infarction (interruption ofblood supply to a part of the heart, causing heart cells to die);cardiac arrest (failure of the heart to contract effectively); heartfailure (a progressive inability of the heart to supply sufficient bloodflow to meet the body's needs, often but not always due to myocardialinfarction or cardiac arrest); cardiac ischemia reperfusion injury(injury to a tissue due to reperfusion of the tissue with bloodfollowing an ischemic condition); cardiomyopathy (muscle weakness due toe.g. ischemia, drug or alcohol toxicity, certain infections (includingHepatitis C), and various genetic and idiopathic (i.e., unknown)causes); injury due to surgery, and degenerative heart diseases such asconduction disease and congenital defects.

Other examples of muscle disorders that could be treated with thesubject cells, particularly allogeneic cells and/or genetically modifiedautologous cells, include muscular dystrophies such as Duchennedystrophy and Becker muscular dystrophy. Duchenne dystrophy is anX-linked recessive disorder characterized by progressive proximal muscleweakness with destruction and regeneration of muscle fibers andreplacement by connective tissue. Duchenne dystrophy is caused by amutation at the Xp21 locus, which results in the absence of dystrophin,a protein found inside the muscle cell membrane. It affects 1 in 3000live male births. Symptoms typically start in boys aged 3 to 7 yr.Progression is steady, and limb flexion contractures and scoliosisdevelop. Firm pseudohypertrophy (fatty and fibrous replacement ofcertain enlarged muscle groups, notably the calves) develops. Mostpatients are confined to a wheelchair by age 10 or 12 and die ofrespiratory complications by age 20. Becker muscular dystrophy is a lesssevere variant, also due to a mutation at the Xp21 locus. Dystrophin isreduced in quantity or in molecular weight. Patients usually remainambulatory, and most survive into their 30s and 40s.

Other particular examples of muscle disorders that could be treated withthe subject cells, particularly allogeneic cells and/or geneticallymodified autologous cells, include the non-dystrophic myopathies such ascongenital and metabolic myopathies, including glycogen storage diseasesand mitochondrial myopathies. Congenital myopathies are a heterogeneousgroup of disorders that cause hypotonia in infancy or weakness anddelayed motor milestones later in childhood. An autosomal dominant formof nemaline myopathy is linked to chromosome 1 (tropomyosin gene), and arecessive form to chromosome 2. Other forms are caused by mutations inthe gene for the ryanodine receptor (the calcium release channel of thesarcoplasmic reticulum) on chromosome 19q. Skeletal abnormalities anddysmorphic features are common. Diagnosis is made by histochemical andelectron microscopic examination of a muscle sample to identify specificmorphologic changes.

Mitochondrial myopathies range from mild, slowly progressive weakness ofthe extraocular muscles to severe, fatal infantile myopathies andmultisystem encephalomyopathies. Some syndromes have been defined, withsome overlap between them. Established syndromes affecting muscleinclude progressive external ophthalmoplegia, the Kearns-Sayre syndrome(with ophthalmoplegia, pigmentary retinopathy, cardiac conductiondefects, cerebellar ataxia, and sensorineural deafness), the MELASsyndrome (mitochondrial encephalomyopathy, lactic acidosis, andstroke-like episodes), the MERFF syndrome (myoclonic epilepsy and raggedred fibers), limb-girdle distribution weakness, and infantile myopathy(benign or severe and fatal). Muscle biopsy specimens stained withmodified Gomori's trichrome stain show ragged red fibers due toexcessive accumulation of mitochondria. Biochemical defects in substratetransport and utilization, the Krebs cycle, oxidative phosphorylation,or the respiratory chain are detectable. Numerous mitochondrial DNApoint mutations and deletions have been described, transmitted in amaternal, nonmendelian inheritance pattern. Mutations in nuclear-encodedmitochondrial enzymes occur.

Glycogen storage diseases of muscle are a group of rare autosomalrecessive diseases characterized by abnormal accumulation of glycogen inskeletal muscle due to a specific biochemical defect in carbohydratemetabolism. These diseases can be mild or severe. In a severe form, acidmaltase deficiency (Pompe's disease), in which 1,4-glucosidase isabsent, is evident in the first year of life and is fatal by age 2.Glycogen accumulates in the heart, liver, muscles, and nerves. In a lesssevere form, this deficiency may produce proximal limb weakness anddiaphragm involvement causing hypoventilation in adults. Myotonicdischarges in paraspinal muscles are commonly seen on electromyogram,but myotonia does not occur clinically. Other enzyme deficiencies causepainful cramps after exercise, followed by myoglobinuria. The diagnosisis supported by an ischemic exercise test without an appropriate rise inserum lactate and is confirmed by demonstrating a specific enzymeabnormality.

Channelopathies are neuromuscular disorders with functionalabnormalities due to distuRBance of the membrane conduction system,resulting from mutations affecting ion channels. Myotonic disorders arecharacterized by abnormally slow relaxation after voluntary musclecontraction due to a muscle membrane abnormality.

Myotonic dystrophy (Steinert's disease) is an autosomal dominantmultisystem disorder characterized by dystrophic muscle weakness andmyotonia. The molecular defect is an expanded trinucleotide (CTG) repeatin the 3″ untranslated region of the myotonin-protein kinase gene onchromosome 19q. Symptoms can occur at any age, and the range of clinicalseverity is broad. Myotonia is prominent in the hand muscles, and ptosisis common even in mild cases. In severe cases, marked peripheralmuscular weakness occurs, often with cataracts, premature balding,hatchet facies, cardiac arrhythmias, testicular atrophy, and endocrineabnormalities. Mental retardation is common. Severely affected personsdie by their early 50s.

Myotonia congenita (Thomsen's disease) is a rare autosomal dominantmyotonia that usually begins in infancy. In several families, thedisorder has been linked to a region on chromosome 7 containing askeletal muscle chloride channel gene. Painless muscle stiffness is mosttroublesome in the hands, legs, and eyelids and improves with exercise.Weakness is usually minimal. Muscles may become hypertrophied. Diagnosisis usually established by the characteristic physical appearance, byinability to release the handgrip rapidly, and by sustained musclecontraction after direct muscle percussion.

Familial periodic paralysis is a group of rare autosomal dominantdisorders characterized by episodes of flaccid paralysis with loss ofdeep tendon reflexes and failure of muscle to respond to electricalstimulation. The hypokalemic form is due to genetic mutation in thedihydropyridine receptor-associated calcium channel gene on chromosome1q. The hyperkalemic form is due to mutations in the gene on chromosome17q that encodes a subunit of the skeletal muscle sodium channel(SCN4A).Subjects suffering from muscular disorders e.g., acute cardiacischemia, degenerative heart diseases such as ischemic cardiomyopathy,conduction disease, and congenital defects, or injury due to surgery,etc. could especially benefit from regenerative tissue therapies.

Diseases other than those of the musculature may similarly be treated byregenerative tissue therapy that employs lineage-restricted cellsproduced by the subject methods. For example, diseases of the centralnervous system (CNS) or the peripheral nervous system (PNS) may betreated by such therapy. For example, for the treatment of Parkinson'sdisease, dopaminergic neurons may be transiently induced to divide,giving rise to neural progenitors (i.e. mitotic cells of the neurallineage) or neural precursors (post-mitotic cells of the neural lineage,i.e. following exit from mitosis) that may be transferred into thesubstantia nigra of a subject suffering from Parkinson's disease.Alternatively, the neural progenitors or neural precursors may beinduced to differentiate into dopaminergic neurons ex vivo, and thentransferred into the substantia nigra or striatum of a subject sufferingfrom Parkinson's disease. Alternatively, dopaminergic neurons of thesubstantia nigra of a subject suffering from Parkinson's disease may beinduced to transiently divide in situ. Descriptions of post-mitoticdifferentiated neurons, neuronal progenitor and precursor cells, and howto culture these cells are have been described in the art. Otherdiseases and disorders of the nervous system that may benefit from thesubject methods include Alzheimer's Disease, ALS, disorders of olfactoryneurons, a disorder of spinal cord neurons, a disorder of peripheralneurons, and other disorders due to injury or disease.

For the treatment of multiple sclerosis, spinal cord injury, or otherdisorder of the Central Nervous System in which enhancing myelination isdesirable to treat the disorder, oligodendrocytes may be transientlyinduced to divide, giving rise to oligodendrocyte progenitors (MPCs) oroligodendrocyte precursors (PMIs), which are then transferred to asubject suffering from a demyelinating condition of the CNS, e.g.Multiple sclerosis, etc. or other condition wherein it is desirable toenhance myelination, e.g. spinal cord injury, etc., at the site whereenhanced myelination is desired. Alternatively, the oligodendrocyteprogenitors or oligodendrocyte precursors may be induced todifferentiate into oligodendrocytes ex vivo, and then transferred intothe subject suffering from the MS, spinal cord injury, etc., at the sitewhere enhanced myelination is desired. Alternatively, oligodendrocytesof a subject suffering from the MS, spinal cord injury, etc. may beinduced to transiently divide in situ at the site where enhancedmyelination is desired. Descriptions of post-mitotic differentiatedoligodendrocytes, oligodendrocyte progenitors, and oligodendrocyteprecursors, and how to culture these cells are described in Dugas, J. etal. (2006) J Neurosci. 26:10967-10983 and US Application No.20090258423, the disclosures of which is incorporated herein byreference.

In other examples, pancreatic islet cell progenitor (MPC) or precursor(PMI) cells derived from post-mitotic differentiated pancreatic isletcells may be transplanted into a subject suffering from diabetes (e.g.,diabetes mellitus, type 1), see e.g., Burns et al., (2006) Curr. StemCell Res. Ther., 2:255-266. Descriptions of post-mitotic differentiatedcells of the pancreas, i.e. islet cells, the progenitor and precursorcells of that lineage, and how to culture these cells are described inU.S. Pat. No. 6,326,201, the disclosure of which is incorporated hereinby reference.

Hepatic progenitor cells or post-mitotic differentiated hepatic cellsderived from post-mitotic differentiated hepatic cells are transplantedinto a subject suffering from a liver disease, e.g., hepatitis,cirrhosis, or liver failure.

In some instances, it will be desirable to regenerate tissue withlineage-restricted cells that were produced from post-mitoticdifferentiated cells of allogeneic tissue, that is, tissue from adifferent host, for example, where the disease conditions result fromgenetic defects in tissue-specific cell function. Where the dysfunctionarises from conditions such as trauma, the subject cells may be isolatedfrom autologous tissue, and used to regenerate function. Autologouscells may also be genetically modified, in order to correct diseaseconditions results from genetic defects. Alternatively, where thedysfunction arises from conditions such as trauma, post-mitoticdifferentiated cells may be transiently induced to divide in situ,giving rise to lineage-restricted cells that will differentiate andincorporate into the injured tissue.

As alluded to above, genes may be introduced into the subjectlineage-restricted cells that have been produced ex vivo for a varietyof purposes, e.g. to replace genes having a loss of function mutation,provide marker genes, etc. Alternatively, vectors are introduced thatexpress antisense mRNA or ribozymes, thereby blocking expression of anundesired gene. Other methods of gene therapy are the introduction ofdrug resistance genes to enable normal progenitor cells to have anadvantage and be subject to selective pressure, for example the multipledrug resistance gene (MDR), or anti-apoptosis genes, such as bcl-2.Various techniques known in the art may be used to introduce nucleicacids into the lineage-restricted cells, e.g. electroporation, calciumprecipitated DNA, fusion, transfection, lipofection, infection and thelike, as discussed above. The particular manner in which the DNA isintroduced is not critical to the practice of the invention.

To prove that one has genetically modified the subjectlineage-restricted cells, various techniques may be employed. The genomeof the cells may be restricted and used with or without amplification.The polymerase chain reaction; gel electrophoresis; restrictionanalysis; Southern, Northern, and Western blots; sequencing; or thelike, may all be employed. The cells may be grown under variousconditions to ensure that the cells are capable of maturation to all ofthe myeloid lineages while maintaining the ability to express theintroduced DNA. Various tests in vitro and in vivo may be employed toensure that the potential of the cells to differentiate into a cell of aparticular lineage has been maintained.

The lineage-restricted cells may be used as a therapy to treat disease(e.g., a genetic defect). The therapy may be directed at treating thecause of the disease; or alternatively, the therapy may be to treat theeffects of the disease or condition. Lineage-restricted cells producedby the ex vivo methods above may be transferred to, or close to, aninjured site in a subject, that is, delivered/administered locally; orthe cells can be introduced to the subject in a manner allowing thecells to migrate, or home, to the injured site. The transferred cellsmay advantageously replace the damaged or injured cells and allowimprovement in the overall condition of the subject. In some instances,the transferred cells may stimulate tissue regeneration or repair.

The lineage-restricted cells may be administered in any physiologicallyacceptable excipient, where the cells may find an appropriate site forregeneration and differentiation. The cells may be introduced byinjection, catheter, or the like. The cells may be frozen at liquidnitrogen temperatures and stored for long periods of time, being capableof use on thawing. If frozen, the cells will usually be stored in a 10%DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells may beexpanded by use of growth factors and/or feeder cells associated withprogenitor cell proliferation and differentiation.

The cells of this invention can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration. Choiceof the cellular excipient and any accompanying elements of thecomposition will be adapted in accordance with the route and device usedfor administration. The composition may also comprise or be accompaniedwith one or more other ingredients that facilitate the engraftment orfunctional mobilization of the cells. Suitable ingredients includematrix proteins that support or promote adhesion of the cells.

The subject methods are useful for both prophylactic and therapeuticpurposes. Thus, as used herein, the term “treating” is used to refer toboth prevention of disease, and treatment of a pre-existing condition.The treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient, is a particularly important benefit provided bythe present invention. Such treatment is desirably performed prior toloss of function in the affected tissues; consequently, the prophylactictherapeutic benefits provided by the invention are also important.Evidence of therapeutic effect may be any diminution in the severity ofdisease. The therapeutic effect can be measured in terms of clinicaloutcome or can be determined by immunological or biochemical tests.

The dosage of the therapeutic formulation will vary widely, dependingupon the nature of the condition, the frequency of administration, themanner of administration, the clearance of the agent from the host, andthe like. The initial dose can be larger, followed by smallermaintenance doses. The dose can be administered as infrequently asweekly or biweekly, or more often fractionated into smaller doses andadministered daily, semi-weekly, or otherwise as needed to maintain aneffective dosage level.

The number of administrations of treatment to a subject may vary.Introducing the lineage-restricted cells into the subject may be aone-time event; but in certain situations, such treatment may elicitimprovement for a limited period of time and require an on-going seriesof repeated treatments. In other situations, multiple administrations ofthe cells may be required before an effect is observed. The exactprotocols depend upon the disease or condition, the stage of the diseaseand parameters of the individual subject being treated, and can bereadily determined by one of ordinary skill in the art.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thecompositions of the invention, e.g. an agent that transiently inhibitsthe activity of a pocket protein family member and an agent thattransiently inhibits the activity of ARF. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

EXAMPLE Materials and Methods

Mice and primary myoblast preparation. Rosa26-CreER^(T2) RB^(lox/lox)mice bred and maintained as described in (Viatour, P., et al. (2008)Cell Stem Cell 3(4): 416-428) were crossed to mice carrying aCre-responsive β-galactosidase reporter allele (Ventura, A., et al.(2007) Nature 445(7128): 661-665). The hind leg muscles of 6-8 weeks oldgenotyped offspring mice were prepared for primary myoblasts harvest asdescribed (Rando, T. A. and Blau, H. M. (1994) The Journal of CellBiology 125(6): 1275-1287). Primary myoblasts after harvest wereselected in F10 media (Gibco) supplemented with 20% FBS (Omegascientific), 2.5 ng/mL bFGF (Promega), and 1% Pen-Strep (Gibco) for aweek on collagen (Sigma) coated plates.

Cell culture. C2C12 mouse myoblasts were cultured at 10% CO₂ 37° C. inDMEM HG (Gibco) supplemented with 20% FBS+1% Pen-Strep. Myoblasts wereseeded for fusion as described (Pajcini, K. V., et al. (2008) TheJournal of Cell Biology 180(5): 1005-1019) under low serum conditions inDMEM supplemented with 2% HS, on collagen coated plates. DM media wasreplaced every 24 hrs. After primary myoblast harvest and selection,passages were counted once cells were cultured and expanded on growthmedia: DMEM LG+F10 media supplemented with 15% FBS, 1% Pen-Strep and1.25 ng/mL bFGF, and plated on collagen coated plates. Primary myoblastswere seeded for fusion under low serum conditions in DMEM LG, 2% HS 1%Pen-Strep at 6×10⁵ cells per 6 cm plate and for sparse, single-celldifferentiation at 5×10⁴ cells per 6 cm plate. Cell seeding numbers wereadjusted depending on the surface area of different plating platformsfrom the 6 cm standards described above.

Cloning and vector construction. pLE-myog3R-GFP retroviral vector wasconstructed by subcloning the myogenin promoter elements driving GFPexpression from peGFPN1-hmyg by digestion at NotI/EcoR0109 into pLE-GFPretroviral vector backbone at XhoI/NaeI sites, after removal of CMV-GFPcassette by blunt cloning (T4 DNA pol and CIP treatment). Both forwardand reverse insertion orientation were tested as described in FIG. 5,with reverse (3R) orientation showing the highest fidelity ofmyogenin-GFP coexpression. pMIG retrovirus vector encoding for the humanRB cDNA (Sage, J., et al. (2000) Genes & development 14(23): 3037-3050)as well as all other vectors employed were transfected into ecotropicphoenix cells using FuGENE 6 (Roche). Cells were infected with viralsupernatants containing polybrene (5 μg ml⁻¹) and centrifuged for 30 minat 2,000 g.

siRNA silencing and semi-quantitative RT-PCR (sqRT-PCR).RNA-interference was carried out using small-interfering RNAs (siRNAs)duplexes designed then screened for specific and effective knockdown oftarget genes. Duplexes employed for experiments shown in this articledesigned for p16/19 sense sequence AGGUGAUGAUGAUGGGCAAUU (SEQ ID NO:11)and p19ARF sense sequence GCUCUGGCUUUCGUGAACAUG (SEQ ID NO:12) orordered directly as ON-TARGETplus siRNA RB1 (J-047474-06) fromThermo/Dharmacon. For control transfections non-targeting siRNA#1(D-001810-01-05) and siGlo-Green were purchased from Thermo/Dharmacon.Transfections of siRNA duplexes, resuspended in siRNA buffer (Dharmacon)were carried out after differentiation of myocytes or after fusion ofmyotubes at 48-72 hrs in DM with silmporter transfection reagent(Millipore) as per manufacturer's instructions with final siRNAconcentration at 100-200 nM. Transfection mix was added to cells aftersupplemented to differentiation media for 12 hrs. RNA was harvested fromC2C12 or primary cells in GM or DM by RNeasy mini kit (Qiagen) and 200ng of total RNA was used in semi-quantitative RT-PCR analysis withSuperscript III One-Step RT-PCR (invitrogen). Primers designed for genestested are listed below all sequences are in 5′-3′ orientation:

RB set 1: For:  (SEQ ID NO: 13) GAGGAGAATTCTGTGGGCCAGGGCTGTG; Rev: (SEQ ID NO: 14) GTACGAGCTCGAGCCGCTGGGAGATGTT product size 1368 bp.RB set 2:  For:  (SEQ ID NO: 15) CAGGCTTGAGTTTGAAGAAATTG  Rev: (SEQ ID NO: 16) ATGCCCCAGAGTTCCTTCTTC  product size 168 bp. p16/19: For:  (SEQ ID NO: 17) CGCCTTTTTCTTCTTAGCTTCA  Rev:  (SEQ ID NO: 18)AGTTTCTCATGCCATTCCTTTC  product size 220 bp. p19ARF:  For: (SEQ ID NO: 19) CCCACTCCAAGAGAGGGTTT  Rev:  (SEQ ID NO: 20)AGCTATGCCCGTCGGTCT  product size 465 bp. Anillin:  For:  (SEQ ID NO: 21)GCGTACCAGCAACTTTACCC  Rev:  (SEQ ID NO: 22) GGCACCAAAGCCACTAACAT product size 202 bp. AuroraB:  For:  (SEQ ID NO: 23)TCGCTGTTGTTTCCCTCTCT  Rev:  (SEQ ID NO: 24) GATCTTGAGTGCCACGATGA product size 388 bp. Survivin:  For:  (SEQ ID NO: 25)CATCGCCACCTTCAAGAACT  Rev:  (SEQ ID NO: 26) AGCTGCTCAATTGACTGACG product size 360 bp. MRF4:  For:  (SEQ ID NO: 27) GGCTGGATCAGCAAGAGAAG Rev:  (SEQ ID NO: 28) CCTGCTGGGTGAAGAATGTT  product size 317 bp.Myogenin:  For:  (SEQ ID NO: 29) TCCAGTACATTGAGCGCCTA  Rev: (SEQ ID NO: 30) GGGCTGGGTGTTAGCCTTAT  product size 470 bp. MHC:  For: (SEQ ID NO: 31) TGAGAAGGAAGCGCTGGTAT  Rev:  (SEQ ID NO: 32)TCTGCAATCTGTTCCGTGAG  product size 588 bp. M-CK:  For:  (SEQ ID NO: 33)GATCTTCAAGAAGGCTGGTCAC  Rev:  (SEQ ID NO: 34) CAATGATTGGACTTCCAGGAG product size 428 bp. GAPDH:  For:  (SEQ ID NO: 35) CACTGAGCATCTCCCTCACA Rev:  (SEQ ID NO: 36) TGGGTGCAGCGAACTTTATT  product size 122 bp

Annealing temperature ranged from 58-62° C. and cycles from 23(Myogenin) to 31 (p19ARF) with most products visible at 26 to 28 cycles.For RNA loading control 50 ng of total RNA was run with GAPDH primersand visualized at 22 cycles.

BrdU analysis. 5-bromo-2′-deoxy-uridine (BrdU) labeling and detectionkit (Roche) was employed on C2C12 myotubes and primary myocytes andmyotubes. BrdU labeling reagent was added to the cells with fresh DMmedia for 12 hrs after the treatments outlaid in each of the schemes inFIG. 1 and FIG. 2. Following labeling the cells were fixed and detectedfor immunofluorescence as per manufacturer's instructions, andco-stained for other proteins as described below.

Western analysis. Cells were lysed at room temperature in lysis buffer(50 mM Tris pH 7.5, 10 mM MgCl₂, 0.3 M NaCl, 2% IGEPAL). Lysates werecleared by centrifugation 5 min at 6000 rpm at 4° C., and proteinquantification was determined by Bradford assay (Bio-Rad).Immunoblotting was conducted using NuPage system from invitrogen.Samples were loaded in 10% Bis-Tris pre-cast gels, resolved by runningwith MES or MOPS SDS buffer, and transferred at 4° C. onto Immobilon-Pmembranes (Amersham). Membranes were blocked in 5% milk-PBS-T thenincubated with antibodies against RB (BD) diluted 1/250 or RB (Sage lab)1/1000; p19ARF (abcam) 1/1000; Survivin (Cytoskeleton Inc) 1/1000;AuroraB (Cytoskeleton Inc.) 1/500; MHC (Chemicon) 1/500; adult MHC (Blaulab) 1/50; M-CK (Novus) 1/500; Myogenin (BD) 1/500; and GAPDH as proteinloading control diluted 1/10000 (Santa Cruz biotech). Membranes werethen incubated with HRP-conjugated secondary antibodies (1/5000) (Zymed)and detected by ECL or ECL⁺ detection reagents (Amersham). Whennecessary, membranes were stripped by incubating at 50° C. for 45 minand then 1 hr at RT in stripping buffer (100 mM 2-mercaptoethanol, 2%SDS, 62 mM Tris, pH7).

Immunofluorescence. C2C12 myotubes and primary myocytes or myotubesseeded densely for fusion or sparsely for differentiation as describedabove were fixed and permeabilized as per manufacturer's instructionswhen co-staining with BrdU or with 1.5% paraformaldehyde 15 min at roomtemperature (RT) then permeabilized with 0.3% Triton-PBS 10 min at RT.All fixed cells were blocked with 20% goat serum for 30 min at RT or 8hrs at 4° C. Primary antibodies for myosin heavy chain (MHC) (Chemicon)diluted 1/75 in blocking buffer or adult MHC diluted 1/20 were incubatedat RT for 30 min. Primary antibodies for myogenin-RBt (Santa Cruzbiotech) 1/100 or myogenin-Ms (BD) 1/250, incubated at RT for 1 hr.Primary antibodies for GFP-RBt (Invitrogen) 1/500 incubated at RT for 1hr. Primary antibodies for survivin (Cytoskeleton Inc.) 1/250 incubatedfor 1 hr at RT. Primary antibodies for Ki67 (Dako) diluted 1/100 andincubated for 8 hrs at 4° C. Secondary antibodies (Invitrogen) Alexa-488GtaMs or GtaRBt and Alexa-546 GtaMs or GtaRBt and Alexa-546 GtaRat werediluted 1/500 and incubated with appropriate primary combinations for 45min at room temperature. When co-staining after BrdU labeling, secondaryantibody for BrdU was FITC-aMs diluted 1/40 and incubated 30 min at 37°C. Nuclear staining of cells with Hoechst 33258 (Sigma) diluted 1/5000and incubated at room temperature for 15 min. Cells were imaged withZeiss Axioplan2 using 40× water immersion objective, Zeiss Axiovert200M, or Zeiss Observer Z1 using NeoFluar 10× or LD Plan NeoFluar 20×objectives while ORCA-ER C4742-95; Hamamatsu Photonics, or Axiocam MRmcameras were used to capture image. Openlab 5.0.2, Volocity 3.6.1(Improvision), and PALM Robo V4 (Zeiss) were the software used for imageacquisition. Images were composed and edited in Photoshop CS (Adobe).Background was reduced using contrast adjustments and color balance wasperformed to enhance colors. All modifications were applied to theentire image.

FACS sorting. Cells were harvested from culture dishes after 0.05%trypsin treatment, centrifuged and resuspended in FACS buffer (PBS+2%GS+2 mM EDTA), and kept on ice until analysis. Cells were analyzed andsorted using a FACSVantage SE (BD Biosciences), with the DIVA analysissoftware. Dead cells were gated out by staining with Propidium Iodide (1μg/ml), and cells were sorted for GFP expression at low pressure topreserve cell viability. Double sort was carried out in order to obtaina purity of 99% viable cells. In the second sort, cells were sorteddirectly in GM or DM as indicated and then seeded in different platforms(microwells or PALM duplex dishes).

PALM LPC. Primary myoblasts were sparsely seeded for differentiation in50 mm or 35 mm laminin (Roche) coated Duplexdishes (Zeiss). 72-96 hrsafter DM switch and upon verification of GFP expression by direct nativefluorescence, myocytes were treated as described in FIG. 6. Duplexdishes were equilibrated by allowing media to flow in between themembrane layers via permeabilization of the top PEN membrane by LPCfunction at least 24 hrs before cell capture. Laser ablation was carriedout after stage calibration, laser focus and optical focus calibrationas per manufacturer's instructions, in Zeiss Observer Z1 invertedmicroscope outfitted with PALM Microbeam (Zeiss). GFP-myogeninexpression was verified for every myocyte selected by directimmunofluorescence with X-CITE series 120 EXFO. Ablation was carried outthrough 20× LD Plan-NeoFluar objective, which permits a 3-5 μm widthlaser track, and membranes ranging from 50 to 150 μm ² in area werecatapulted by LPC, burst point of which was selected to be at least 10μm from the myocyte. Media was removed from the Duplexdish until only afilm of moisture covered the plate; in 50 mm dishes, this condition canbe obtained by retaining only 500 μL of media. Ablated membranes werecatapulted by LPC bursts into Roboarm SingleTube Capture II receptacle(500 μL eppendorf tube cap) with 80 μL of media. Membrane/myocytecapture was verified after capture by direct observation of the capturedreceptacle. Total volume of the receptacle was transferred into 12 or24-well collagen coated plates, where captured myocytes were cultured inconditioned growth media (cGM), which was harvested from activelydividing myoblasts and filtered through a 0.2 μm filter.

Immunocytochemistry. 10 days after injection, TA muscles were dissectedand immersed in PBS/0.5% EM-grade PFA (Polysciences) for 2 h at roomtemperature followed by overnight immersion in PBS with 20% sucrose at4° C. Section staining and image analysis was performed as described inPajcini, K. V., et al. (2008) The Journal of cell biology 180(5):1005-1019.

Timelapse microscopy. Primary myoblasts were seeded for fusion in 6-wellcollagen coated plates. After 72 hrs in DM, primary myoblasts weretreated with TAM. 24 hours later, i.e. at 96 hrs in DM, one set ofprimary myotubes were treated with 200 nM p16/19si for 12 hrs, at whichpoint transfection mix was washed and cells were placed in fresh DMmedia. 12 hrs later, primary myotubes were imaged using Zeiss Axiovert200M equipped with timelapse apparatus CTI-Controller 3700 Digital;Tempcontrol 37-2 digital; scanning stage Incubator XL 100/135 (PECON).Frames were captured every 10 min for a total of 50 hrs, encompassingdays 5 and 6 during primary myotube fusion and maturation. Images wereacquired and analyzed using Volocity 3.6.1 (Improvision). Growth medium(GM); Differentiation medium (DM).

Results

Suppression of RB by siRNA induces S-phase re-entry in C2C12 myotubes.The myoblast cell line C2C12 is a model system for studying muscledifferentiation in vitro. In low serum differentiation medium (DM),confluent C2C12 myoblasts exit the cell cycle and fuse with one anotherto form multinucleated muscle cells (myotubes), which express muscleproteins and are contractile. We developed a protocol to transientlyexpress siRNA molecules in more than 60% of myotubes using thesilmporter reagent (FIG. 9). To determine the duration and efficiency ofsiRNA treatment, C2C12 myotubes were treated with siRNA-duplexes againstRB (RBsi) (FIG. 1A-B). Semi-quantitative RT-PCR of RB transcript levelsindicated that transient suppression of RB mRNA was strongest up to 48hrs post-transfection, after which time RB levels began to recover, butnever reached the same level of expression seen in control-siRNA(Mocksi) treated myotubes at 96 hrs. (FIG. 1B). Suppression of RBprotein levels in myotubes was confirmed by western blotting. Total celllysates were harvested from proliferating myoblasts and fromdifferentiated myotubes treated with Mocksi or RBsi. Treatment with RBsiresulted in a gradual loss of RB protein (FIG. 1E) to 50% of the levelof control RB in differentiated myotubes (FIG. 1F).

To determine if S-phase re-entry in C2C12 myotubes occurred aftersuppression of RB, we labeled Mocksi and RBsi-treated myotubes withBrdU, as indicated in the scheme in FIG. 1A. FIG. 1D showsrepresentative images. Myotubes were stained for myosin heavy chain(MHC) along with BrdU to enhance morphological identification and toconfirm differentiation. BrdU-labeled myonuclei were observed insideMHC⁺ myotubes only after RBsi treatment (middle and lower panels). Wefound a marked change in myotube morphology 48 hrs after transfection ofRBsi (FIG. 1D bottom panels). After suppression of RB, myotubes losttheir compact elongated structure and characteristically alignedmyonuclei. Cell shape no longer resembled a myotube and nucleiaggregated in clusters, many of which were BrdU-positive. S-phasere-entry was dependent on the dose of RBsi used and the percentage ofBrdU⁺ myonuclei doubled from 25% to 52% as the siRNA concentration wasincreased from 100 to 200 nM (FIG. 1C). In all of the followingexperiments, unless stated otherwise, the 200 nM concentration of siRNAwas used. The percentage of myotubes that contained any BrdU-positivenuclei was analyzed in a separate set of experiments and found to be 25%(FIG. 10F), which is explicable because myotubes with Brdu-positivenuclei often had large clusters of nuclei that underwent DNA synthesis,probably reflecting the successful transfection of a fraction of thecells in the culture (FIG. 9A). FIG. 1F shows representative images ofBrdU incorporation together with MHC immunostaining to confirmdifferentiation. We found a marked change in myotube morphology 72 hrafter transfection of Rbsi (FIG. 1D, bottom) from a compact elongatedstructure with characteristic linear nuclear alignment to an amorphousstructure, with nuclei aggregated in clusters, many of which were BrdUpositive. These data confirm that transient suppression of RB issufficient to induce S-phase re-entry in differentiated C2C12 myotubenuclei, and show that the extent of this effect depends on theconcentration of RBsi used.

Both RB and p19ARF must be suppressed for S-phase re-entry in primarymyotubes. The majority of the data that suggests that suppressing RBalone is sufficient for cell cycle re-entry in mature mammalian myotubeshas been obtained in experiments using the C2C12 cell line. Althoughuseful for studying various aspects of muscle cell differentiation andfusion, C2C12 cells, like other cell lines, have acquired mutations thatpermit their immortalization. Therefore, we considered it critical touse primary cells to assess the role of RB in maintaining thepost-mitotic state. We harvested primary myoblasts capable ofdifferentiation in culture, as previously described (Rando, T. A. andBlau, H. M. (1994) The Journal of Cell Biology 125(6): 1275-1287), fromthe hind leg muscles of Rosa26-CreER^(T2) RB^(lox/lox) mice crossed tomice carrying a Cre-responsive β-galactosidase reporter allele. In thesemice, Cre expression and RB excision is dependent on tamoxifen (TAM)induction (FIG. 10A-B). Primary myoblasts were used at low passageduring which time cell shape remained compact and uniform. In primarymyotube cultures a single 24 hr treatment with 1 μM TAM was sufficientto reduce RB expression (FIG. 10C). A time course of RB expressionindicates that both transcript and protein levels drop substantially by96 hrs following TAM treatment (FIG. 2C and FIG. 10D).

We analyzed S-phase re-entry in primary myotube cultures after loss ofRB expression by BrdU-labeling according to the scheme in FIG. 2A. Bycontrast to C2C12 myotubes, in primary myotubes regardless of whether RBwas knocked-down by RBsi (FIG. 2B) or excised by Cre expression (FIG. 2Dtop panels), BrdU-labeling of myonuclei was rare (FIG. 2G). BrdUlabeling in MHC⁺ myotubes was assayed in greater than 1500 nuclei inrandomly selected fields. Notably, in the representative images in FIGS.2B and 2D, BrdU⁺ nuclei are clearly present and detectable, but thesenuclei are not within MHC⁺ myotubes as shown in merged IF images. Thus,regardless of the method of RB suppression, loss of RB in primarymyotubes is not sufficient for cell cycle re-entry.

Disruption of RB function by mutation or by hyperphosphorylation inresponse to mitogenic signals leads to the accumulation of E2Ftranscription factor activity, and activation of ARF (DeGregori, J., etal. (1997) Proceedings of the National Academy of Sciences USA 94(14):7245-7250; Lowe, S. W. and Sherr, C. J. (2003) Current Opinion inGenetics & Development 13(1): 77-83). ARF, in turn, serves to blockinappropriate cycling. Induction of ARF was accompanied by a mildincrease in baseline levels of apoptosis, but the majority of myotubesremained robust and viable. Apoptosis was rarely observed insiRNA-treated cells in which p16/p19 was reduced (FIG. 13).

In contrast, C2C12 myotubes do not express p19ARF in response to RBsuppression, as would be expected in a cell line that has bypassed cellcycle regulation by the Ink4a locus during immortalization (FIG. 2E). Agenomic deletion in the shared exon 2 region of the ink4a locus wasconfirmed by PCR (FIG. 2F). To test whether the suppression of the Ink4agene products in RB-deficient primary myotubes would permit cell cyclere-entry, we designed siRNA duplexes that target the shared exon 2region of Ink4a mRNA (p16/19si), and the ARE-specific exon 1β region forknockdown of p19ARF (p19ARFsi). Primary myotubes were treated with TAMor RBsi, then 24 hrs later transfected with p16/19si, p19ARFsi or both.Loss of RB and p19ARF in myotubes was verified by western analysis (FIG.2C). When labeled with BrdU following TAM and p16/19si treatment asdescribed in the scheme in FIG. 2A, myonuclei incorporated BrdU inMHC-positive myotubes (FIG. 2D). Quantification of the BrdU labelingindicated that 45.7±7.2% of myonuclei enter S-phase in TAM and p16/19sitreated myotubes, a marked increase over baseline values observed inmyotubes treated only with TAM or p16/19si respectively (FIG. 2H).Different combinations of siRNAs that exclusively knockdown p19ARF orboth Ink4a gene products in TAM treated cells did not yieldsignificantly different results (FIG. 2H). For subsequent experiments weused the p16/19si (exon 2) because it gave the strongest suppression ofp19ARF. Our data shows that robust S-phase re-entry in differentiatedprimary mammalian myotubes occurs only after combined suppression ofboth RB and p19ARF.

Upregulation of mitotic and cytokinetic components in RB andp16/19-defficient myonuclei. To determine if S-phase re-entry in PMmyonuclei marked the initiation of the mitotic process in differentiatedmultinucleated myotubes, we analyzed control or TAM and p16/19si treatedprimary myotube nuclei for the induction of expression of mitotic andcytokinetic proteins. We investigated the expression patterns andlocalization of AuroraB and survivin, two important components of thechromosome passenger complex (CPC), which controls chromosome andspindle structure, kinetochore attachment and chromosome segregation. Wealso analyzed the mRNA expression of anillin, a structural proteinimportant in the organization and stability of the cleavage furrowduring cytokinesis.

Anillin and each of the CPC components mentioned above are activelyexpressed in primary proliferating mononucleated myoblasts, but theirmRNA and protein levels drop precipitously once myotubes form (FIG.3A-B). Once RB was excised in differentiated primary multinucleatedmyotubes after TAM-treatment, anillin, AuroraB and survivin mRNA levelsall rose, despite high levels of p19ARF expression (FIG. 3A). However,protein levels for AuroraB and survivin attain levels comparable tothose of growing myoblasts only after concomitant suppression of RB andp19ARF (FIG. 3B). To verify that upregulation of CPC components occurredspecifically in myonuclei that had entered S-phase, primary myoblastswere labeled with BrdU for 12 hrs then fixed and stained for BrdU andsurvivin. Dividing primary myoblasts in growth medium are positive forboth BrdU and survivin, with the latter marking the cleavage furrowbetween two dividing cells (FIG. 12 arrowhead). Differentiated,control-treated myotubes do not have myonuclei that express survivin,but those treated with TAM and p16/19si exhibit clustered BrdU⁺myonuclei, and it is these same myonuclei that upregulate survivinexpression (FIG. 3C bottom panels). Quantification of the stainingresults indicated that nearly 80% of the myonuclei that had re-enteredS-phase also upregulated survivin (FIG. 3D). In the vast majority ofBrdU⁺ myonuclei, survivin staining did not localize in the cleavagefurrow or to any particular nuclear compartment, but was diffuselydistributed throughout the nucleus. Taken together, our data show thatin primary multinucleated myotubes deficient in both RB and ARF, DNAsynthesis is followed by upregulation of proteins involved in chromosomesegregation and cytokinesis.

Dedifferentiation accompanies S-phase re-entry in primary muscle cells.Differentiated muscle cells exhibit characteristic changes in expressionof phenotypic markers such as MHC and creatine kinase (M-CK) thataccompany the profound changes observed in their morphology andpost-mitotic state; see, for example, Charge, S. B. and Rudnicki, M. A.(2004) Physiological Reviews 84(1): 209-238. We investigated the role ofthe RB and Ink4a gene products in sustaining expression of thedifferentiated phenotype by analyzing morphology and expression ofmarkers of differentiation. In primary myoblasts in kept in growthmedium or undergoing differentiation (DM), the cellular morphology andexpression of MHC were analyzed at various time points, with or withoutdeletion of RB and with or without suppression of p16/19. The data showthat over time, cells which are treated with TAM first undergo formationof mature myotubes with strong expression of MHC, followed at later timepoints by a strong decrease in MHC expression after loss of expressionof RB, but only if ARF is also suppressed (FIG. 4A). In cells treatedonly with TAM, not only does the morphology remain similar to controlmyotubes, but MHC staining remains strong (FIG. 4A).

In primary myotubes, protein analysis of cell lysates revealed that somemyogenic protein levels are moderately sensitive to the loss of RBalone. A moderate decline in MHC and myogenin levels was observed inprimary myotubes following treatment with TAM only (FIG. 4B). However,RBsi treatment caused only a slight drop in myogenin protein levels inprimary myotubes (FIG. 4D third lane). Suppression of both RB andp19ARFconsistently led to reduced protein levels of all myogenicproteins tested. For example, in the case of alpha tubulin, anotherprotein with specific roles in differentiation of muscle cells, thecombined suppression of RB and p16/19 resulted in a marked decrease inprotein levels (FIG. 4 F). This occurred in parallel with the increasedexpression of the mitotic proteins AuroraB and survivin (FIG. 4B).Quantification of MHC levels revealed a marked decrease in expression ofthis protein after suppression of RB and p19ARF when normalized tolevels in control cells in DM for the same period of time (FIG. 4C).These findings, as well as a drop in the expression levels of MRF4,another transcriptional regulator of late differentiation, are supportedby semi-quantitative RT-PCR analysis (FIG. 15C).

Morphological deterioration of myotube structure correlated with BrdU⁺staining in primary myotubes as in C2C12 myotubes, however, only if bothRB and p16/19 were suppressed. FIG. 4D exemplifies the structuralcollapse of primary myotubes after TAM and p16/19si treatment. Whilecontrol primary myotubes retained their elongated morphology and nuclearorganization, BrdU⁺ myotubes collapsed into amorphous multinucleatedsyncytial structures, highlighted by the clustering of myonuclei. Whilenot all of the myonuclei are in S-phase, structural integrity of themyotube has been lost likely due to the lack of maintenance of myotubenuclear protein domains. Myotubes were visualized by time-lapsemicroscopy after treatment with TAM and Mocksi (Video 1) or TAM andp16/19si. A time-lapse comparison shows that complete morphologicalcollapse of myotube structure only takes place after loss of both the RBas well as Ink4a gene products. Despite extensive structuraldifferences, the RB and ink4a deficient primary myotubes do not die ordetach faster than Mocksi-treated myotubes, and retain their motilityand membrane activity, such as filopodia and lamellapodia protrusions.

Post-mitotic differentiated myocytes are capable of proliferation aftersuppression of RB and p16/19. The extent of muscle differentiation canbe characterized based on the serial expression of muscle regulatorytranscription factors. MyoD and Myf5 are early and characteristic ofcycling myoblasts, whereas late transcription factors include myogeninand MRF4 which are expressed in post-mitotic differentiated myocytes andmyotubes. Based on this transcription factor timecourse, we sought todevelop a system in which we could study cycling and dedifferentiationin prospectively isolated populations derived from individualdifferentiated muscle cells. We infected low passageRosa26-CreER^(T2)RB^(lox/lox) primary myoblasts with a retroviralexpression vector in which GFP expression is under the control of themyogenin promoter (pLE-myog3R-GFP). To verify the fidelity of myogeninand GFP co-expression, pLE-myog3R-GFP myoblasts were sparsely seeded ingrowth medium or differentiation medium for 72 hours and stained formyogenin and GFP. IF analysis clearly showed that GFP and myogeninexpression were upregulated in the differentiating (DM3) myocytepopulation (FIG. 5A). Quantification of IF data indicated that only 0.9%of the cells are GFP-positive in growth conditions, likely representingspontaneous differentiation due to physical contact. In differentiatedcultures, GFP expression occurred in 27±2.0% of the cells (FIG. 5Bi).When individual cells were analyzed by microscopy, 98.4% of the cellswith detectable GFP also expressed detectable levels of myogenin.High-throughput analysis of GFP expression in myoblasts and myocytes wasalso performed by fluorescence activated cell sorting (FACS), whichshowed that 35% of the cell population expressed GFP after 3 days indifferentiation medium, while only 1.8% of cells in growth medium wereGFP⁺ (FIG. 5D). Thus pLE-myog3R-GFP infection of primary myoblastsallows for reliable identification of myogenin-expressing populations ofcells by means of GFP expression.

Myoblasts seeded at low density will express myogenin and undergoterminal differentiation in the absence of fusion. We took advantage ofthis in vitro property of muscle cells to investigate whether thededifferentiation and cell cycle re-entry observed in differentiatedmultinucleated myotubes could lead to proliferation after suppression ofRB and p19ARF in differentiated mononucleated myocytes. The followingexperiments were performed using individual pLE-myog3R-GFP muscle cells,in order to follow the fate of single cells. First, sparsely seededprimary muscle cells, maintained in DM for 72 hrs, were sorted on thebasis of GFP expression as depicted by the gated population in FIG. 5C.To determine whether the differentiated muscle cells were capable ofproliferation, the FACS sorted population was cultured in conditionedgrowth medium (cGM) for up to 48 hrs and then stained (FIG. 5E toppanels) for GFP and Ki67, a nuclear marker of cellular proliferation(Scholzen and Gerdes 2000). Only 2.3% of the sorted population had Ki67positive nuclei. In contrast, cells of the sorted population treatedwith TAM 24hrs prior to FACS sorting, and with p16/19si 12 hrs afterFACS sorting (FIG. 5E bottom panels) exhibited Ki67 nuclear staining in25% of the sorted population 48 hrs after culture in cGM (FIG. 5F).Second, as an alternative method to suppress RB and p16/19, siRNAduplexes against RB and p16/19 were used to transiently knockdownexpression of both. Analyses to determine the ideal dosage and method ofsiRNA application showed that most efficient knockdown occurred aftertandem treatment of primary muscle cells with RBsi, a 12 hr recoveryperiod, followed by treatment with p16/19si. This double-knockdown (DKD)treatment protocol resulted in comparable results to the TAM andp16/19si treatment, while a combination of siRNA duplexes appliedsimultaneously was not as efficient in silencing the expression ofeither gene (FIG. 10E). DKD treatment of FACS sorted GFP⁺ cells,resulted in 8.6% Ki67⁺ nuclei. That the frequency of DNA synthesis waslower in DKD treated cells than in those treated with TAM and p16/19sivalue was expected given the lower efficiency of a knockdown compared toTAM treatment for RB suppression. The data from these two types ofexperiments show at the single cell level, that differentiatedmyogenin-expressing myocytes, like myotubes, efficiently enter S-phaseonly after suppression of both RB and Ink4a gene expression.

Since mono-nucleated, differentiated myocytes enter S-phase followingsilencing of RB and p16/19, we assayed whether these cells were capableof completing the cell-cycle and proliferating. We reasoned that ifmyocytes could divide, they would give rise to clones. However, to ruleout cell migration and definitively show that a postmitotic myocytedivided, single-cell resolution and clonal analysis was critical.Accordingly, to assess the proliferative potential of myocytes, we firstFACS-purified cells twice in order to isolate individual differentiatedGFP⁺ myocytes which were then sorted directly into microarrays ofhydrogel wells. Myocytes were first imaged 12 hrs after sorting intomicrowells, at which time they were treated with Mocksi or the firstapplication of DKD treatment (FIG. 17A left panels). Images ofmicrowells were captured at 48, 72 and 96 hrs after the completion ofDKD treatment. Proliferative myocytes were scored as the percentage ofmicrowells that had a minimum of 8 cells at 96 hrs after treatment.Although overall viability of the cells was impaired due to the toxicityof the slmportter used for RNAi delivery, which could not be adequatelyremoved from the microwells, a clear difference was evident in thepercentage of clones generated from myocytes. 6.8% of DKD treatedmyocytes gave rise to clones whereas only 0.6% of Mocksi treated cells(FIG. 17B). Raw data showed that a total of 109 colonies arose from theDKD treated myocytes, but only 10 colonies arose from the mock treatedmyocyte population. Taken together these data support the conclusionthat myogenin-positive myocytes acquire proliferation potential aftersuppression of RB and p16/19 expression.

Clonal expansion and proliferation of individually purified myocytesafter laser microdissection and laser pressure catapulting. We sought toincrease the purity of all of our prospectively isolated cells byevaluating each individual myocyte for differentiation prior to capture.FACS sorting in combination with the hydrogel microwell platformdescribed above can be readily used to monitor single-cell proliferationpotential. However, several disadvantages precluded subsequent analysis:heterogeneity of the sorted population, disruption of cellularmorphology upon detachment from the plate, and lack of post-treatmentmolecular analysis. These obstacles can be overcome by the use ofphotoactivated laser microdissection (PALM) and laser pressurecatapulting (LPC), which enables pure and homogenous individual cellsample preparation without disruption of cell adhesion and morphology,and permits expansion and molecular analysis after colony establishment.See, for example, Stich, M., et al. (2003) Pathology, research andpractice 199(6): 405-409; Schutze, K., et al. (2007) Methods in cellbiology 82: 649-673).

Laser microdissection and pressure catapulting analysis was performedwith myocytes. For this purpose primary myoblasts expressingpLE-myog3R-GFP+ were sparsely seeded and differentiated to become GFP+myocytes and then treated with either mock siRNA (FIG. 6A) or forsuppression of Rb and ARF (FIGS. 5Bi and 5Ci). Individual myocytes wereselected based on their differentiated phenotype including elongatedmorphology evident by phase microscopy (FIG. 6Ai, left) and bright GFPexpression (FIGS. 6Bii and 6Cii, left). Laser microdissection was usedto mark and cut the membrane surrounding prospectively identified singlemyocytes (green lines FIG. 6Bii and FIG. 6Cii). Marking and recordingmembrane shapes allowed for tracking of each membrane and the isolatedcell on its surface. The LPC burst locations that lead to catapultingare indicated by the blue dots in the images in FIG. 6Bii, which alsoserve as a means of identifying the membrane. Typical images obtainedduring the steps of the PALM and LPC isolation process are shown inFIGS. 6Bii and 6Cii. Myocyte morphology before and after membraneablation did not significantly change. Note in the example shown that 72hr after capture, the mocktreated myocyte is still associated with themembrane (FIG. 6Aii, panel 4), and by 96 hr it has left the membrane butstill exists as a single adherent cell, although it was cultured inconditioned growth medium since the time of capture (FIG. 6Aii, panel5). Similarly, in individual myocytes treated for reduction of Rb andARF, morphology remained intact during the cutting and catapulting(FIGS. 6Bii and 6Cii, left 3 panels). However, in contrast tomock-treated myocytes, reduction of Rb (TAM or siRNA) and ARF (siRNA)led to cell division and colony formation in the immediate vicinity ofthe membrane (FIG. 6Bii, fourth panel from left, FIG. 6Cii, fourth panelfrom left). Additional examples of single GFP+ myocyte laser capture areshown in FIG. 18.

In five independent PALM LPC isolation experiments, an analysis of atotal of 250 membranes verified that without RB and p16/19 suppressionnot a single myogenin-GFP⁺ myocyte captured divided to produce a colony.In contrast, TAM and p16/19si treated myocytes produced 34 colonies, afrequency of 14% colony formation (FIG. 6C). Transient DKD treatedmyocytes formed colonies at a frequency of 8%, which although lower,corresponds to the frequency of colony formation observed for myocytesobtained by FACS and microwell expansion above (FIG. 13B). One technicalproblem with live-cell capture by PALM LPC is desiccation of the cellsduring the LPC phase of cell isolation, which can decrease viability.Finding and documenting the GFP expressing myocytes by microscopy takestime. To overcome this problem, we used FACS to identify and sortmyocytes expressing GFP, which were then treated as indicated in thescheme in FIG. 19A. Images of the captured control or TAM and p16/19sitreated myocytes are shown which provide further evidence that thecontrol treated myocytes fail to produce colonies in conditioned growthmedium (FIG. 19B), while myocytes in which RB and Ink4a genes weresilenced (FIG. 19C), exhibited a frequency of 28% colony formation (FIG.6D). Taken together these results strongly support a role for RB andInk4a in the maintenance of myogenic differentiation, as theirsuppression leads to division and expansion of myogenin-expressingdifferentiated myocytes.

Redifferentiation of captured myocyte colonies after exposure to lowserum.

Dedifferentiated muscle cells in axolotls are thought capable ofproliferation and contribution to regenerating muscle. To determine ifdedifferentiated, actively dividing mammalian myocytes are capable ofredifferentiation after expansion, we exposed captured cells todifferentiation conditions. After LPC capture, TAM and p16/19si-treatedmyocytes proliferated rapidly in culture in GM, and most of the cellslost myogenin expression after 72 hrs, evidenced by lack of GFP (FIG.20B). However, even when exposed to DM for 3 days, thesededifferentiated myocytes continued to proliferate, by comparison withuntreated LPC captured myoblasts, which had started to fuse by this time(FIG. 20A). Indeed, the TAM and p16/19si treated population never fused,but instead continued to divide until cellular aggregates were observedby 6 days in differentiation medium (FIG. 20B lower panels). This lackof differentiation is expected because the cells have genetically lostRB expression, which is necessary for differentiation.

In contrast to the TAM-treated captured myocytes, RB suppression in DKDcaptured myocytes was transient (FIG. 1B). Four colonies derived fromsingle DKD-treated captured myocytes were expanded, split, exposed to DMfor four days and then assayed for muscle markers, either by microscopyor biochemically (see scheme in FIG. 6). The DKD colonies spanned thespectrum of differentiation potential, the two extreme ends of which areshown in the top panels of FIG. 7A and FIG. 7C. One colony (DKDcap1)continued to proliferate despite being in DM while another (DKDcap2)differentiated and fused. Heterogeneity in the behavior of capturedcells after transient knockdown is expected, as each derived fromextensive proliferation.

Protein analysis of the DKD captured colonies supports a function for RBin successful redifferentiation, since DKDcap1 cells have lost RBexpression, while the colonies that readily fused and differentiated(DKDcap4 and DKDcap2) expressed high levels of RB in DM (FIG. 7B).p19ARF expression in DKDcap1 was high regardless of media conditions, asexpected for highly proliferative cells lacking RB. In contrast, in thecolonies that fused to produce large myotubes, the expecteddownregulation of p19ARF was observed in DM. Expression of myogenin andMHC provided further confirmation of the myogenic potential of theredifferentiated myocytes. Protein levels of myogenin and MHC increasedupon exposure of DKDcap4 and DKDcap2 to DM (FIG. 7B), while myogenin andGFP expression, hallmarks of the cells at the time of capture wereupregulated only in redifferentiated DKDcap2 myotubes (FIG. 7C). Therelative changes in expression patterns of RB, Ink4a and myogenicproteins by the DKDcap2 colony and by captured myoblasts were similar(FIG. 21B).

Based on these observations, we reasoned that if RB was re-introducedinto the TAM and p16/19si captured cells, redifferentiation and fusionshould occur. Thus, we infected a subset of captured colonies with apMIG retrovirus vector expressing the human RB cDNA. We monitored theTAMcap myoblast colonies after infection with pMIG-RB or control pMIGretrovirus and determined that differentiation and fusion occurred inthose colonies that re-expressed RB (FIG. 7A lower panels). Althoughdedifferentiating myotubes did not reactivate expression of Pax-7(possibly because of limited duration of viability in culture) isolateddedifferentiated clones did, as evidenced by Western analysis, thusfulfilling this criterion of a proliferating primary myoblast (FIG. 7E).Analysis of RB protein levels harvested in growth and differentiationmedia from control or pMIG-RB infected TAMcap colonies showed theexpected increase in RB protein following pMIG-RB retroviral infection,which occurred in parallel with upregulation of the myogenic proteins,myogenin and MHC (FIG. 7B). While p19ARF levels never acquired theexpression pattern observed in the DKDcap colonies, survivin levelsdecreased in the TAMcap1 colony following pMIG-RB infection (FIG. 7B,lane 6). The myogenic potential of TAMcap colonies infected with pMIG-RBwas also verified by IF for MHC (FIG. 7D). Together, these datademonstrate that dedifferentiated myocytes are capable of successfulredifferentiation in vitro after capture and expansion, and that thisprocess is dependent upon expression of RB (FIG. 8B).

Captured and expanded myocytes are capable of contribution to muscle invivo. Finally, we tested whether PALM LPC isolated, expanded myocytescould contribute to existing muscle in vivo. 1.5×10⁵ DKD deriveddedifferentiated myocytes (DKDcap1 and DKDcap2) were injected into thetibialis anterior (TA) of NOD/SCID mice. Myogenin-GFP expression wasused to track the injected cells, as this marker was reliably detectedin vitro upon differentiation (FIG. 7C). Ten days after injection,DKDcapl cells, which exhibited no fusion in vitro, proliferatedexcessively in vivo and caused severe disruption of the muscle lamininnetwork at the site of injection (FIG. 22, top panels). On the otherhand, DKDcap2 cells readily fused to the existing muscle fibers andcaused no disruption of the laminin network (FIG. 22, bottom panels).

Visualization of the myofibers in which DKDcap2 cells fused wasdifficult due to weak GFP expression. While in vitro imaging of GFP⁺ inredifferentiated myotubes was facilitated by fusion of multiplemyogenin-GFP cells, in vivo contribution of a few injected cells to anon-GFP myofiber significantly larger than a culture-derived myotubeexplains the weak GFP signal observed in vivo. To overcome this problem,we co-infected TAMcap colonies with pMIG-RB and pLE-GFP retrovirus, thusproviding these cells with a copy of RB as well as bright constitutiveGFP expression. Control TAMcap1 cells, which received control pMIG andpLE-GFP infections, proliferated excessively in vivo (FIG. 8A toppanels). By contrast, TAMcap1 cells with re-introduced RB expression,efficiently fused to existing muscle fibers, brightly labeling them withGFP, without any apparent proliferation and without disruption of theexisting laminin network (FIG. 8A bottom panels). We conclude that if RBexpression is adequately restored dedifferentiated myocytes are capableof redifferentiation and incorporation by fusion to existing muscle invivo.

Discussion

The molecular basis for the extraordinary disparity between mammals andcertain lower vertebrates, such as newts, axolotls and zebrafish, intheir capacity to regenerate injured or amputated tissues remains amajor unresolved biological question. Mammalian regeneration of a muscleoccurs only if that muscle is replaced after mincing or grafting ofsmall muscles, or when chemical agents spare stem cells. Experiments ofthis type indicate that significant architectural remodeling can occurin lieu of scarring but that the extent of regeneration is limited bythe amount of replaced or surviving muscle. Although muscle stem cellscan account for some degree of regeneration, they do not seem tosuffice. Indeed, to date there is no evidence that the extensiveregeneration of entire muscles seen in urodeles can be achieved by anyknown mammalian mechanism when a significant mass of tissue is removedand not replaced. This limited regenerative potential is in part due toa combination of an excessive demand for cell proliferation at the siteof injury in order to replace lost tissue, and an inhibition ofarchitectural remodeling by fibrosis. A possible basis for the failureof regeneration in mammals, which has fascinated scientists forcenturies, is a capacity, which newts have retained and mammals havelost: dedifferentiation.

Does dedifferentiation endow urodeles with the regenerative capabilitiesthat mammals lack? This question underscores the need for novel insightsinto the molecular mechanisms of dedifferentiation in order to discoverwhat is missing in mammals. In urodeles, compelling evidence fromstudies of skeletal muscle suggests that dedifferentiation is a majormode of tissue regeneration. Dedifferentiation involves two processes,which are separable and independent: muscle cell fragmentation intoindividual mononuclear cells, and cell cycle re-entry followed byproliferation. In mammalian myotubes produced using the immortalizedcell line, C2C12, overexpression of transcriptional factors present inthe blastema such as msx1 or twist, or exposure to small molecules thatdisrupt the cytoskeleton, such as myoseverin, have been reported toresult in myotube fragmentation. In the dedifferentiation studiesreported here, fragmentation of muscle cells was not observed, which isin good agreement with reports showing that the fragmentation process innewts is independent of cell cycle re-entry (Velloso, C. P., et al.(2000) Differentiation; research in biological diversity 66(4-5):239-246). In addition, since the trigger and mechanism for musclefragmentation and cellularization in urodeles remains unknown, it iscurrently not possible to determine if a similar pathway exists inmammals. Understanding how fragmentation occurs and delineating thededifferentiation mechanisms are complementary but distinct goals inmuscle regeneration biology.

Molecular regulation of dedifferentiation by RB and ARE Our decision tosuppress

RB in experiments directed at elucidating the mechanisms underlyingmuscle dedifferentiation was based on evidence in newts that RBcoordinates muscle cell cycle entry in response to damage. In mammals asin newts, cell cycle regulation by RB is dynamic and controlledprimarily by its phosphoryation state. There is also a wealth of datafirmly establishing the tumor suppressor RB as a necessary player in theorchestration of mammalian muscle cell differentiation, includingevidence for a dual role in both muscle cell cycle progression and exit.Indeed, RB null mice die before birth and lack differentiated muscles.Furthermore, RB has also been shown to act not only as a cell cycleregulator, but also to impact differentiation and tissue specific geneexpression directly by binding histone deacetylase 1 (HDAC1) andpromoting activation of muscle genes such as MyoD. Once differentiationoccurs, this state is stably maintained, at least in mammals. Thisstability is underscored by the inability to reverse differentiationsimply by inactivating RB in primary differentiated mammalian musclecells. Indeed, studies reporting otherwise have been confounded by theuse of immortalized cell lines such as C2C12, which we show here do notexpress the ink4a products. Loss or suppression of RB leads only tomoderate dedifferentiation, as demonstrated by the reduced accumulationof myogenin and MHC (FIG. 4). In fact, as shown in this report, it isremarkable how little phenotypic change occurs in primary differentiatedskeletal muscle cells when RB is suppressed.

The minimal impact of RB absence alone on muscle dedifferentiationsuggested that maintenance of mammalian differentiation is ensured by aseparate mechanism. Whereas the RB pathway is intact in lowervertebrates, we reasoned that another component that is absent inregeneration competent vertebrates would be a good candidateregeneration suppressor in mammals. This line of thought led us to focuson the ink4a locus and ARF in particular. Unlike RB, inactivation of ARFalone in knockout mice has no apparent effect on differentiation. Inagreement with these reports, we found that ARF alone had no effect onmuscle differentiation or dedifferentiation. However, concomitantinactivation of RB and ARF caused extensive dedifferentiation.Differentiated myotubes exhibited robust DNA synthesis and activation ofmitotic proteins upon acute loss of RB and p19ARF, suggesting that theseproteins are nodal points for intrinsic control of muscle cell cyclereentry. The profound loss of architectural integrity and downregulationof myogenin, MRF-4, MHC and M-CK upon suppression of both RB and p19ARFfurther suggests that the two together are potent stabilizers of thedifferentiated state. Notably, alternative approaches that inducecycling by altering growth factor signaling and regeneration inmammalian cardiac muscle cells produce a very moderate effect whencompared to regenerating urodele muscle. We speculate that ARF mayinhibit robust cycling and regeneration in these settings as well.

Our findings support the hypothesis that tumor suppression mediated bythe RB and ink4a loci arose at the expense of regeneration. Bothp16ink4a and p19ARF have been recently shown to contribute to thedecline in regenerative potential of multiple tissues during aging byaffecting stem cell self-renewal. Our study suggests that the ink4alocus has an additional negative impact on tissue regeneration, i.e.,suppression of cell cycle re-entry and dedifferentiation. The remarkablecombined effect of acute RB and ARF loss strongly suggests (i) thatcontinuous expression of RB itself has an important function inmaintaining the differentiated state and (ii) that the maintenance ofthe differentiated state in mammals depends on complementary activitiesof RB and ARF. These findings are explicable in view of the known needfor continuous regulation of differentiation as well as the documentedfunctions of RB and ARF in preventing inappropriate cycling as tumorsuppressors.

Single cell analyses of dedifferentiation. Bulk cultures do not allow adefinitive assessment that a given cell has divided. The cellularcomplexity and rapid developmental changes observed in the blastema hashindered analysis of dedifferentiation at the single-cell level inurodele regeneration. In mammalian muscle culture systems in whichS-phase re-entry was observed, the persistence of cells at earlierstages of differentiation cannot be ruled out; see, for example, Gu, W.,et al. (1993) Cell 72(3): 309-324; Schneider, J. W., et al. (1994)Science (New York, N.Y. 264(5164): 1467-1471;and Blais, A., et al.(2007) The Journal of cell biology 179(7): 1399-1412). In addition,continuous timelapse monitoring and single cell analysis are essentialsince reports of division of differentiated muscle cells could be theresult of cell migration. To overcome these problems we employed (i)dynamic single-cell tracking of myocytes isolated in microwells by timelapse microscopy and (ii) isolation of single myocytes by PALM lasercapture microscopy. These single cell studies clearly demonstrated thatcell cycle entry and expansion of individual differentiated postmitoticmyocytes occurs after RB and p19ARF loss. We further demonstrated theregenerative potential of dedifferentiated myocytes by inducingredifferentiation. A subset of captured colonies produced by transientinactivation of RB and p19ARF were exposed to differentiation medium inculture and fused to form myotubes. Additionally, in myocytes that hadirreversibly lost RB expression due to Cre-mediated excision,reintroduction of RB by retroviral delivery not only induced myotubeformation and muscle gene expression in vitro, but also resulted infusion and regeneration of damaged myofibers in vivo with typicalarchitecture and no evidence of the tumorigenic characteristics of cellsthat did not receive RB. These findings suggest that transientinactivation of the two tumor suppressors could yield dedifferentiatedcells with extensive regenerative potential as depicted in the diagramin FIG. 8B.

We capitalized on evolutionary differences to genetically modify themammalian cell-cycle regulatory pathways to more closely mimic thosefound in lower vertebrates. Our results reveal that it is possible toderive regenerative cells from differentiated, post-mitotic muscle inaddition to classically defined stem cells. Skeletal muscle cells canalternate between a differentiated, post-mitotic state and aproliferative, regenerative state, retaining the essentialcharacteristics of their cell type of origin during the regenerativecycle. Our experiments implicate ARF in the suppression of regenerationin mammalian cells by impeding dedifferentiation. Thus, a combination ofinterventions to transiently inactivate the RB pathway in aphysiological manner while suppressing ARF may be employed to maximize amammalian regenerative response.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

That which is claimed is:
 1. A method of producing lineage-restrictedcells (LRCs) from a post-mitotic differentiated cell (PMD) of the samelineage, comprising: contacting a PMD with an effective amount of anagent that transiently inhibits the activity of a pocket protein and aneffective amount of an agent that transiently inhibits the activity ofthe cyclin-dependent kinase inhibitor 2A alternate reading frame protein(ARF) under conditions sufficient to permit the PMD to transientlydivide to produce progeny, wherein said progeny are LRCs of the samelineage as the PMD.
 2. The method of claim 1, wherein said PMDdedifferentiates prior to dividing.
 3. The method of claim 1, whereinsaid PMD is a myocyte.
 4. The method of claim 3, wherein said myocyte isselected from the group consisting of a cardiomyoctyte, a smooth musclemyocyte, a skeletal myocyte, and myofiber.
 5. The method of claim 1,wherein said pocket protein is retinoblastoma protein (RB).
 6. Themethod of claim 5, wherein said agent that transiently inhibits RBactivity is a nucleic acid, a polypeptide, or a small molecule.
 7. Themethod of claim 1, wherein said agent that transiently inhibits ARFactivity is a nucleic acid, a polypeptide, or a small molecule.
 8. Themethod of claim 1, wherein about 10% of PMDs of a population that arecontacted are induced to transiently divide.
 9. The method of claim 1,wherein the PMD are from an individual with a disease.
 10. The method ofclaim 1, wherein the individual is alive.
 11. The method of claim 1,wherein the individual is a cadaver.
 12. The method of claim 1, whereinthe method is effected in vivo.
 13. The method of claim 1, wherein themethod is effected ex vivo.
 14. The method of claim 1, wherein saidmethod further comprises transferring said progeny LRCs to conditionsthat promote differentiation, wherein the population that is produced isa population of PMDs of the same lineage as the PMD that was contactedin said contacting step.
 15. The method of claim 14, wherein saidtransferring is effected by transplanting said progeny into a subject.16. The method of claim 15, wherein said subject is in need of tissueregeneration therapy.
 17. A method of screening a candidate agent for aneffect on a disease condition, comprising: producing lineage-restrictedcells (LRCs) from a post-mitotic differentiated cell (PMD) from anindividual with said disease condition by the method of claim 1,transferring said LRCs to conditions that promote differentiation toproduce a differentiated population of cells, contacting saiddifferentiated population of cells with a candidate agent, and comparingthe viability and/or function of the cells in said differentiatedpopulation to the viability and/or function of differentiated cells notcontacted with said candidate agent; wherein enhanced viability and/orfunction of the cells in the differentiated population contacted withsaid candidate agent as compared to a differentiated population notcontacted with said candidate agent indicates that the candidate agentwill have an effect on said disease condition.
 18. The method of claim17, wherein said disease condition is a muscle disorder.
 19. The methodof claim 18, wherein the muscle is smooth muscle, skeletal muscle, orcardiac muscle.
 20. The method of claim 17, wherein said diseasecondition is a nervous system disorder.
 21. The method of claim 20,wherein the nervous system disorder is Parkinson's Disease, Alzheimer'sDisease, ALS, a disorder of olfactory neurons, a disorder of spinal cordneurons, or a disorder of peripheral neurons.
 22. The method of claim17, wherein said individual is alive.
 23. The method of claim 17,wherein said individual is a cadaver.
 24. A method of screening acandidate agent for toxicity to a human comprising: producinglineage-restricted cells (LRCs) from a post-mitotic differentiated cell(PMD) from a healthy individual by the method of claim 1, transferringsaid LRCs to conditions that promote differentiation to produce adifferentiated population of cells, contacting said differentiatedpopulation of cells with a candidate agent, and comparing the viabilityand/or function of the cells in said differentiated population to theviability and/or function of differentiated cells not contacted withsaid candidate agent; wherein a decrease in viability and/or function ofthe cells in the differentiated population contacted with said candidateagent as compared to a differentiated population not contacted with saidcandidate agent indicates that the candidate agent is toxic to a human.25. The method of claim 24, wherein the PMD is a hepatocyte.
 26. Themethod of claim 24, wherein the function of the cells is assess byassessing a cytochrome P450 panel.
 27. A method of producinglineage-restricted cells (LRCs) from post-mitotic differentiated cells(PMDs) in a tissue in a subject, comprising: contacting PMDs of saidtissue in vivo with an effective amount of an agent that transientlyinhibits the activity of a pocket protein and an effective amount of anagent that transiently inhibits the activity of the cyclin-dependentkinase inhibitor 2A alternate reading frame protein (ARF), wherein thecontacted cells are induced to transiently divide in situ so as toproduce LRCs of the lineage of the PMD's lineage.
 28. The method ofclaim 27, wherein said PMD dedifferentiates prior to dividing.
 29. Themethod of claim 27, wherein said PMDs are myocytes.
 30. The method ofclaim 29, wherein said myocytes are selected from the group consistingof cardiomyoctytes, smooth muscle myocytes, skeletal myocytes, andmyofibers.
 31. The method of claim 27, wherein said pocket protein isretinoblastoma protein (RB).
 32. The method of claim 27, wherein saidagent that transiently inhibits RB activity is a nucleic acid, apolypeptide, or a small molecule.
 33. The method of claim 27, whereinsaid agent that transiently inhibits ARF activity is a nucleic acid, apolypeptide, or a small molecule.
 34. The method of claim 27, whereinsaid agent that transiently inhibits RB activity and said agent thattransiently inhibits ARF activity are administered locally to thetissue.
 35. The method of claim 27, wherein said subject is a subject inneed of tissue regeneration therapy.
 36. A kit for use in transientlyinducing post-mitotic differentiated cells to transiently divide,comprising an agent that transiently inhibits the activity of a pocketprotein and an agent that transiently inhibits the activity of ARF.